Allergy inhibitor compositions and kits and methods of using the same

ABSTRACT

Compositions and kits for inhibiting an allergic response against an allergenic protein are disclosed. The compositions comprise a eukaryotic cell expression vector containing nucleotide sequences encoding an allergenic protein or a polypeptide that comprises an antigenic epitope of said allergenic protein; and an allergenic protein or a polypeptide that comprises an antigenic epitope of the allergenic protein. The kits comprise a first container which comprises a eukaryotic cell expression vector containing nucleotide sequences encoding an allergenic protein or a polypeptide that comprises an antigenic epitope of the allergenic protein and a second container which comprises an allergenic protein or a polypeptide that comprises an antigenic epitope of said allergenic protein. Compositions and kits for inhibiting an allergic response against an flea allergenic protein; a feline allergenic protein; a canine allergenic protein; a dust mite allergenic protein; a peanut allergenic protein; a Japanese cedar allergenic protein; and a blomia tropicalis allergenic protein are disclosed. Methods if using such compositions and kits are also disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to Chinese application number 200510132381.X filed Dec. 23, 2005, which is incorporated herein byreference.

FIELD OF THE INVENTION

The present invention relates to compositions and kits which are usefulto prevent and inhibit allergic reactions against allergens, and tomethods of using such compositions and kits. The present inventionprovides compositions, kits and methods for preventing and inhibitingflea allergy dermatitis, allergic reactions to cat and canine fur ordanders, dust mites, peanuts, Japanese cedar pollen and blomiatropicalis allergen.

BACKGROUND OF THE INVENTION

Allergic reactions to various allergens represent significant healthconcerns, particularly in instances in which the allergic reactioninduces severe reactions and/or allergen induced immediatehypersensitivity (AIH).

Allergy is considered as the consequence of persistent T cell activationdriving pathogenic inflammation against host dermis by specificallergens. Several approaches are used to ameliorate AIH and theseinclude nonspecific immunosuppressive drugs or monoclonal antibodiestargeted to T or B cells (A. J. Van Oosterhout et al., Am. J. Respir.Cell Mol. Biol. 17, 386 (Sep. 1, 1997); P. Proksch et al., J Immunol174, 7075 (Jun. 1, 2005)). However, this situation is compromised aslong term treated recipients can become generally compromised in theirability to fight infections. Redirecting immunity from Th2 type to Th1type has also been demonstrated with limited success (S. Jilek, C.Barbey, F. Spertini, B. Corthesy, J Immunol 166, 3612 (Mar. 1, 2001)). Arecent discovery of T regulatory cells, including the naturallyoccurring thymus derived CD4⁺CD25⁺ Treg cells (I. M. de Kleer et al., J.Immunol 172, 6435 (May 15, 2004); D. Lundsgaard, T. L. Holm, L. Hornum,H. Markholst, Diabetes 54, 1040 (Apr. 1, 2005); M. J. McGeachy, L. A.Stephens, S. M. Anderton, J Immunol 175, 3025 (Sep. 1, 2005); I.Bellinghausen, B. Klostermann, J. Knop, J. Saloga, J Allergy ClinImmunol 111, 862 (Apr. 1, 2003); E. M. Ling et al., Lancet 363, 608(Feb. 21, 2004); and J. Kearley, J. E. Barker, D. S. Robinson, C. M.Lloyd, J. Exp. Med., jem.20051166 (Nov. 28, 2005)), mucosal induced Th3cells and antigen induced CD4⁺CD25⁻ Tr cells have been proposed to beuse as immuno-regulators or suppressors or auto-reactive pathogenesis(H. Fukaura et al., J. Clin. Invest. 98, 70 (Jul. 1, 1996)). Variousapproaches have been explored to induce T regulatory cells to constrainthe auto-reactive T cells. Preferentially, induction of antigen specificT regulatory cells targeted to allergy, asthma and autoimmune diseaseantigens are considered a promising strategy. Several lines of evidencehave indicated that induction of antigen specific regulatory T cell 1(TO) is possible via utility of immatured DCs, suboptimal immunogens orpartial blocking the co-stimulatory molecules in DCs (A. Kumanogoh etal., J Immunol 166, 353 (Jan. 1, 2001); M. K. Levings et al., Blood 105,1162 (Feb. 1, 2005); and S. K. Seo et al., Nat Med 10, 1088 (Oct. 1,2004)). All these approaches are done either in vitro or in experimentalconditions. Induction of Tr cells that can inhibit antigen specific Tcells' function in vivo by co-inoculating antigen-matched DNA andprotein antigens as co-administered vaccines (H. Jin et al., Virology337, 183 (Jun. 20, 2005)).

The chief characteristic of the non-host flea is that it is ahematophagic parasite that may be found in the body of any mammalian oravian species of animal. Ctenocephalides felis is a parasite that occursmainly in cats and dogs, while Ctenocephalides canis is limited todomestic dogs and feral dogs. Flea allergy dermatitis (FAD) is the mostfrequently seen skin ailment in cats and dogs. FAD results when a fleaparasite bites and its saliva serves as an irritant and elicits anallergic reaction. The location of the bite appears red, swollen,irritated and itching. Often the animal will scratch at the bite withits paws, causing the wound to turn into a skin ulceration and elicitingfurther bacterial and fungal infections. This poses a great danger forthe dog or cat and at present no effective pharmacotherapeutic orpreventive methods exist for this disease.

In general, flea allergen refers to the various differently sizedproteins from flea antigens that cause an allergic reaction. In someparts of the literature it is referred to as feline flea salivaallergenic protein FSA1 or Cte f 1. GeneBank AF102502, which isincorporated herein by reference, discloses the nucleotide sequences(SEQ ID NO:1) encoding the FSA1 or Cte f I protein derived from the fleasalivary gland of the Ctenocephalides felis. The 653 nucleotide sequenceincludes coding sequences 1-531 which include coding sequences for thesignal peptide (1-54) and mature protein sequence (55-528). GeneBankAAD17905, which is incorporated herein by reference, discloses the aminisequences (SEQ ID NO:2) of the FSA1 or Cte f I protein derived from theflea salivary gland of the Ctenocephalides felis. including the signalpeptide (1-18) and mature protein sequences (19-176).

The chief feline allergenic protein is Fe1 dI. GeneBank M74953, which isincorporated herein by reference, discloses the amino acid of andnucleotide sequences (SEQ ID NO:3) encoding the Fe1 dI protein derivedfrom the major allergen of the domestic cat. It possesses the secondaryB secretion peptide sequence. This Fe1 d I sequence is 416 bp mRNAincluding the 5′ untranslated region made up of sequences 1-25 and thecoding sequence being sequences 26-292 encoding 88 amino acids (SEQ IDNO:4; GeneBank AAC41617, which is incorporated herein by reference). Thesignal peptide is encoded by 26-79 and the mature protein is encoded by80-289. The 3′ untranslated region is 293-416. GeneBank M74952, which isincorporated herein by reference, discloses the amino acid of andnucleotide sequences (SEQ ID NO:5) encoding the Fe1 dI protein derivedfrom the major allergen of the domestic cat. This Fe1 dI sequence is 410bp mRNA including the 5′ untranslated region made up of sequences 1-7and the coding sequence being sequences 8-286 encoding 92 amino acids(SEQ ID NO:6; GeneBank AAC37318, which is incorporated herein byreference). The signal peptide is encoded by 8-73 and the mature proteinis encoded by 74-283. The 3′ untranslated region is 287-410.

The chief canine allergenic proteins are the salivary lipid promotersCan f1 and Can f2. GeneBank AF027177, which is incorporated herein byreference, discloses the amino acid of and nucleotide sequences (SEQ IDNO:7) encoding the Can f1 protein derived from the salivary lipocalinproteins of the major allergen of the domestic dog. This Can f1 sequenceis 525 bp mRNA encoding 174 amino acids (SEQ ID NO:8; GeneBank AAC48794,which is incorporated herein by reference).

GeneBank AF027178, which is incorporated herein by reference, disclosesthe amino acid of and nucleotide sequences (SEQ ID NO:9) encoding theCan f2 protein derived from the salivary lipocalin proteins of the majorallergen of the domestic dog. This Can f2 sequence is 791 bp mRNAincluding a coding sequence of 195-737 encoding 180 amino acids (SEQ IDNO:10; GeneBank AAC48795, which is incorporated herein by reference).

GeneBank U11695, which is incorporated herein by reference, disclosesthe amino acid of and nucleotide sequences (SEQ ID NO:11) encoding thedust mite allergy source protein antigen Der P1. This Der P1 sequence is1099 bp mRNA including a coding sequence of 50-1012 encoding 180 aminoacids (SEQ ID NO:12; GeneBank AAB60125, which is incorporated herein byreference). The coding sequence includes coding sequences 50-109 whichencode a signal peptide and coding sequences 344-1009 which encodes themature peptide. GeneBank AAB60125 discloses a signal peptide thatincludes amino acids 1-20 and a mature protein that includes amino acids99-320.

GeneBank L77197, which is incorporated herein by reference, disclosesthe amino acid of and nucleotide sequences (SEQ ID NO: 13) encoding thepeanut allergy source protein antigen Ara h II. This Ara h II sequenceis 717 bp sequence encoding 110 amino acids and including a polyA signal562-567.

GeneBank AF059616, which is incorporated herein by reference, disclosesthe amino acid of and nucleotide sequences (SEQ ID NO:14) encoding thepeanut allergy source protein antigen Ara h II. This Ara h 5 sequence is743 bp sequence including a coding sequence of 17-412. GeneBankAAD55587, which is incorporated herein by reference, discloses the 131amino acid protein (SEQ ID NO:15).

GeneBank AB081309, which is incorporated herein by reference, disclosesthe amino acid of and nucleotide sequences (SEQ ID NO: 16) encoding theJapanese cedar (cryptomeria japonica) allergy source antigen Cry j 1.1.This Cry j 1.1 sequence is 1295 bp sequence including a coding sequenceof 62-1186 in which a signal peptide is encoded by 62-124 and the matureprotein is encoded by 125-1183 and a polyA site at 1295. GeneBankBAB86286, which is incorporated herein by reference, discloses the 374amino acid protein (SEQ ID NO:17) including a signal peptide of aminoacids 1-21 and a mature protein of amino acids 22-374.

GeneBank AB081310, which is incorporated herein by reference, disclosesthe amino acid of and nucleotide sequences (SEQ ID NO:18) encoding theJapanese cedar (cryptomeria japonica) allergy source antigen Cry j 1.2.This Cry j 1.2 sequence is 1313 bp sequence including a coding sequenceof 46-1170 in which a signal peptide is encoded by 46-108 and the matureprotein is encoded by 109-1167 and a polyA site at 1313. GeneBankBAB86287, which is incorporated herein by reference, discloses the 374amino acid protein (SEQ ID NO:19) including a signal peptide of aminoacids 1-21 and a mature protein of amino acids 22-374.

GeneBank U59102, which is incorporated herein by reference, disclosesthe amino acid of and nucleotide sequences (SEQ ID NO:20) encoding theblomia tropicalis allergy source protein antigen Blo t 5. This Blo t 5sequence is 537 bp sequence including a coding sequence of 33-437.GeneBank AAD10850, which is incorporated herein by reference, disclosesthe 134 amino acid protein (SEQ ID NO:21).

There remains a need for compositions and methods of preventing andinhibiting the allergic reactions induced by these allergens.

SUMMARY OF THE INVENTION

The present invention relates to compositions for preventing andinhibiting an allergic response against an allergenic protein. Thecompositions comprise:

a) an eukaryotic cell expression vector containing nucleotide sequencesencoding an allergenic protein or a polypeptide that comprises anantigenic epitope of said allergenic protein; and,

b) an allergenic protein or a polypeptide that comprises an antigenicepitope of said allergenic protein.

The present invention provides compositions for preventing andinhibiting an allergic response against an allergenic protein selectedfrom the group consisting of: a flea allergenic protein; a felineallergenic protein; a canine allergenic protein; a dust mite allergenicprotein; a peanut allergenic protein; a Japanese cedar allergenicprotein; and a blomia tropicalis allergenic protein.

The present invention further relates to kits for preventing andinhibiting an allergic response against an allergenic protein. The kitscomprise:

a) a first container comprising a eukaryotic cell expression vectorcontaining nucleotide sequences encoding an allergenic protein or apolypeptide that comprises an antigenic epitope of said allergenicprotein; and

b) a second container an allergenic protein or a polypeptide thatcomprises an antigenic epitope of said allergenic protein.

The present invention provides kits for preventing and inhibiting anallergic response against an allergenic protein selected from the groupconsisting of: a flea allergenic protein; a feline allergenic protein; acanine allergenic protein; a dust mite allergenic protein; a peanutallergenic protein; a Japanese cedar allergenic protein; and a blomiatropicalis allergenic protein.

The present invention further relates to methods of preventing andinhibiting an allergic reaction to an allergenic protein in anindividual. The methods comprise the step or steps of administering tothe individual

a) an eukaryotic cell expression vector containing nucleotide sequencesencoding an allergenic protein or a polypeptide that comprises anantigenic epitope of said allergenic protein; and

b) an allergenic protein or a polypeptide that comprises an antigenicepitope of said allergenic protein.

The present invention provides of preventing and inhibiting an allergicreaction to an allergenic protein in an individual wherein saidallergenic protein is selected from the group consisting of: a fleaallergenic protein; a dust mite allergenic protein; a peanut allergenicprotein; a Japanese cedar allergenic protein; and a blomia tropicalisallergenic protein.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1 a-1 c refers to a Flea allergy model in mice. C57b/6 mice wereprimed twice biweekly with flea antigens or saline as a negativecontrol, challenged with the flea antigens, or PBS as the negativecontrol, histamine as the positive control intradermally. In FIG. 1 a,the local reactions after the skin test were measured at 30 min afterthe challenge. FIG. 1 b shows anti-flea antigens of IgE production. InFIG. 1 c, CD4+ T cell proliferation responses stimulated by fleaantigens in vitro, were tested in mice. Results are representative of atleast three experiments. *P<0.05, compared with naive control groups asindicated.

FIGS. 2 a-2 d show that co-immunization of DNA and protein suppressesthe development of immediate hyper-sensitivity reaction. FIG. 2 a showsdata from skin tests of mice after co-immunization with F or pcDF100+F.FIG. 2 b shows that dose dependent skin responses in mice afterco-immunization with pcDF100+F is displayed. FIG. 2 c shows anti-fleaantigen levels of IgE and IgG1 after induction and treatment. FIG. 2 dshows CD4+ T cell proliferation responses stimulated by fleaantigen-specific in vitro. Results are representative of at least threeexperiments. *P<0.05, compared with V+F and F vaccination groups asindicated.

FIGS. 3 a-3 c show that CD4⁺CD25⁻ T cells are responsible for theobserved suppression. In FIG. 3 a, 5×10⁵ of CD3⁺ T cells were isolatedfrom the spleens of flea antigen immunized mice and added to96-well-plates. At the same time, 1×10⁵ of splenocytes from naive, F,V+F and pcDF100+F immunized mice were also added to the same plate.Similarly, 1×10⁵ non-T cells or T cells, purified CD8⁺, CD4⁺ orCD4⁺CD25″T cells were isolated from the spleens of V+F or pcDF100+Fimmunized mice. These Co-cultures were stimulated with flea antigen (50μg/ml) in the presence of 1×10⁵ bone marrow derived DCs for 48 h invitro. Proliferation was examined by MTS-PMS (Promega) according tomanufactors instructions and stimulation index (SI) was determined bythe formula: counts of flea-antigen stimulated/counts of non-stimulatedcultures). In FIG. 3 b, 1×10⁶ of splenocytes from naive, F, V+F andpcDF100+F immunized mice were adoptively transferred into naive C57mice. Similarly, 1×10⁶ non-T cells or T cells, 1×10⁶ purified CD8⁺,CD4⁺, 5×10⁵ CD4⁺CD25⁻ and CD4⁺CD25⁺T cells were isolated from spleens ofpcDF100+F, V+F, F immunized or naive control mice and were adoptivelytransferred into syngeneic flea-antigen primed mice and skin testresponses were examined. FIG. 3 c shows that co-administration of DNAand protein induce antigen-specific suppression. 1×10⁶CD4⁺CD25⁻ T cellswere isolated from spleens of pcDF100+F, V+F immunized or naive controlmice and were adoptively transferred into naive mice, which were thenimmunized with specific Flea antigen or non-specific OVA protein 24hours after transfer, then CD4⁺ T cells were isolated and theirproliferation was analyzed. Results shown in the figure arerepresentative of two experiments. *P<0.05 compared with V+F and Ftransfers as indicated.

FIGS. 4 a-4 c show that DCs from pcDF100+F co-immunized mice induce Trcells in vitro. FIG. 4 a shows pcDF100+F co-immunization restricted MLRstimulatory activities on DCs. 48 h after immunization, DCs isolatedfrom spleen of mice were used to stimulate T cell proliferation in MLR.T cell proliferation was measured by CFSE activity. Results arerepresentative of one of two respective experiments. *P<0.05 comparedwith V+F and F vaccination groups as indicated. FIG. 4 b shows data fromDCs isolated from spleens of pcDF100+F, V+F, F immunized or naivecontrol mice co-cultured with naive CD4⁺ T cells. The T cells wererestimulated once for two days for 3 cycles, using fresh DCs, and werethen analyzed after each stimulation cycle for IL-10, IL-4 and IFN-γpositive cell numbers, and for the ability to regulated MLR stimulatoryactivities. In FIG. 4 c, MLR was performed with APC from C57 mice and Tcells from Balb/c mice. T cell proliferation was measured by CFSE.Results are representative of two individual experiments. *P<0.05compared with V+F and F vaccination groups as indicated.

FIGS. 5 a-5 c shows dermatological scores data.

FIG. 6 shows results of skin tests before co-immunization.

FIG. 7 shows results of skin tests after co-immunization.

FIG. 8 shows dermatological scores data after co-immunization.

FIGS. 9A-9F show data demonstrating therapeutic effects ofco-immunization on the FAD cats.

FIGS. 10A-10C show data demonstrating therapeutic effects ofco-immunization on the FAD cats.

FIG. 11 shows a map of plasmid pVAX1-K-FSA1.

DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION

The present invention provides compositions, kits and methods whichprevent and inhibit allergic reactions, and allergen induced immediatehypersensitivity. The present invention provides compositions, kits andmethods which prevent and inhibit flea allergy dermatitis, compositions,kits and methods which prevent and inhibit feline allergy, compositions,kits and methods which prevent and inhibit canine allergy, compositions,kits and methods which prevent and inhibit mite allergy, compositions,kits and methods which prevent and inhibit peanut allergy, compositions,kits and methods which prevent and inhibit Japanese cedar allergy andcompositions, kits and methods which prevent and inhibit blomiatropicalis allergy.

The compositions of the invention comprise an allergenic protein or apeptide or protein which comprises an antigenic epitope of theallergenic protein and an expression vector which encodes an allergenicprotein or a peptide or protein which comprises an antigenic epitope ofthe allergenic protein.

The kits of the invention comprise a container that comprises anallergenic protein or a peptide or protein which comprises an antigenicepitope of the allergenic protein and container that comprises anexpression vector which encodes an allergenic protein or a peptide orprotein which comprises an antigenic epitope of the allergenic protein.

The methods of the invention comprise administering the compositions ofthe invention and/or the components of a kit of the invention incombination to an individual who has or is susceptible to allergicreactions, or allergen induced immediate hypersensitivity.

The allergenic protein or a peptide or protein which comprises anantigenic epitope of the allergenic protein present in the compositionor kit and used in the method, and allergenic protein or a peptide orprotein which comprises an antigenic epitope of the allergenic proteinencoded by the expression vector present in the composition or kit andused in the method have amino acid sequence overlap such that they shareepitopes, i.e at least one epitope of the allergenic protein or apeptide or protein which comprises an antigenic epitope of theallergenic protein present in the composition or kit and used in themethod is the same as at least one epitope of the allergenic protein ora peptide or protein which comprises an antigenic epitope of theallergenic protein encoded by the expression vector present in thecomposition or kit and used in the method. In some embodiments, theallergenic protein or a peptide or protein which comprises an antigenicepitope of the allergenic protein present in the composition or kit andused in the method is the same as the allergenic protein or a peptide orprotein which comprises an antigenic epitope of the allergenic proteinencoded by the expression vector present in the composition or kit andused in the method. In some embodiments, the allergenic protein or apeptide or protein which comprises an antigenic epitope of theallergenic protein present in the composition or kit and used in themethod is a fragment of the allergenic protein or a peptide or proteinwhich comprises an antigenic epitope of the allergenic protein encodedby the expression vector present in the composition or kit and used inthe method. In some embodiments, the allergenic protein or a peptide orprotein which comprises an antigenic epitope of the allergenic proteinencoded by the expression vector present in the composition or kit andused in the method is a fragment of the allergenic protein or a peptideor protein which comprises an antigenic epitope of the allergenicprotein present in the composition or kit and used in the method. Insome embodiments, the allergenic protein or a peptide or protein whichcomprises an antigenic epitope of the allergenic protein present in thecomposition or kit and used in the method is a fragment of theallergenic protein or a peptide or protein which comprises an antigenicepitope of the allergenic protein encoded by the expression vectorpresent in the composition or kit and used in the method. In someembodiments, one or both of 1) the allergenic protein or a peptide orprotein which comprises an antigenic epitope of the allergenic proteinpresent in the composition or kit and used in the method and 2) theallergenic protein or a peptide or protein which comprises an antigenicepitope of the allergenic protein encoded by the expression vectorpresent in the composition or kit and used in the method is identical toa naturally occurring protein which is an allergen. In some embodiments,both of 1) the allergenic protein or a peptide or protein whichcomprises an antigenic epitope of the allergenic protein present in thecomposition or kit and used in the method and 2) the allergenic proteinor a peptide or protein which comprises an antigenic epitope of theallergenic protein encoded by the expression vector present in thecomposition or kit and used in the method are identical to a naturallyoccurring protein which is an allergen. In some embodiments, one or bothof 1) the allergenic protein or a peptide or protein which comprises anantigenic epitope of the allergenic protein present in the compositionor kit and used in the method and 2) the allergenic protein or a peptideor protein which comprises an antigenic epitope of the allergenicprotein encoded by the expression vector present in the composition orkit and used in the method is identical to a fragment of a naturallyoccurring protein which is an allergen. In some embodiments, both of 1)the allergenic protein or a peptide or protein which comprises anantigenic epitope of the allergenic protein present in the compositionor kit and used in the method and 2) the allergenic protein or a peptideor protein which comprises an antigenic epitope of the allergenicprotein encoded by the expression vector present in the composition orkit and used in the method are identical to a fragment of a naturallyoccurring protein which is an allergen. In some embodiments, theallergenic protein or a peptide or protein which comprises an antigenicepitope of the allergenic protein present in the composition or kit andused in the method is identical to a fragment of a naturally occurringprotein which is an allergen and the allergenic protein or a peptide orprotein which comprises an antigenic epitope of the allergenic proteinencoded by the expression vector present in the composition or kit andused in the method is identical to a naturally occurring protein whichis an allergen. In some embodiments, the allergenic protein or a peptideor protein which comprises an antigenic epitope of the allergenicprotein present in the composition or kit and used in the method isidentical to a naturally occurring protein which is an allergen and theallergenic protein or a peptide or protein which comprises an antigenicepitope of the allergenic protein encoded by the expression vectorpresent in the composition or kit and used in the method is identical toa fragment of naturally occurring protein which is an allergen.

In some embodiments, the composition or kit includes an allergenicprotein such as a protein from a pathogen, food, environmental factor orirritant. In some embodiments, the composition or kit includes a peptideor protein which includes an antigenic epitope of an allergenic proteinsuch as a protein from a pathogen, food, environmental factors orirritant. Similarly, in some embodiments, the composition or kitincludes an expression vector which encodes an allergenic protein suchas a protein from a pathogen, food or irritant and in some embodiments,the composition or kit includes an expression vector which encodes apeptide or protein which includes an antigenic epitope of an allergenicprotein such as a protein from a pathogen, food, environmental factor orirritant.

In some embodiments, an allergenic protein or peptide or protein whichincludes an antigenic epitope of an allergenic protein that is encodedby the expression vector is identical to the allergenic protein orpeptide or protein which includes an antigenic epitope of an allergenicprotein included in the composition or kit. In some embodiments, anallergenic protein or peptide or protein which includes an antigenicepitope of an allergenic protein that is encoded by the expressionvector is different from the allergenic protein or peptide or proteinwhich includes an antigenic epitope of an allergenic protein included inthe composition or kit. In some embodiments, the peptide or proteinincluded in the composition is the allergenic protein. In someembodiments, the peptide or protein included in the composition is afragment of the allergenic protein. In some embodiments, the peptide orprotein encoded by the expression vector is the allergenic protein. Insome embodiments, the peptide or protein encoded by the expressionvector is a fragment of the allergenic protein. According to theinvention, the methods comprise administering the compositions inamounts sufficient to suppress the allergic reaction against theallergenic protein when the individual is subsequently exposed to suchprotein.

In some embodiments, the present invention provides inhibitors for fleaallergy dermatitis. The flea allergy dermatitis inhibitor of the presentinvention comprises a eukaryotic cell expression vector containing fleasalivary allergenic protein (such as felis salivary antigen 1 (FSA1 orCte f1)) or a peptide or protein that comprises an antigenic epitope ofsuch allergenic protein, in combination with a flea salivary allergenicprotein (such as felis salivary antigen 1 (FSA1 or Cte f1)) or a peptideor protein that comprises an antigenic epitope of such allergenicprotein.

In some embodiments, the present invention provides inhibitors forfeline allergy. The feline allergy inhibitor of the present inventioncomprises a eukaryotic cell expression vector containing felineallergenic protein (such as Fe1 dI) or a peptide or protein thatcomprises an antigenic epitope of such allergenic protein, incombination with a feline allergenic protein (such as Fe1 dI) or apeptide or protein that comprises an antigenic epitope of suchallergenic protein.

In some embodiments, the present invention provides inhibitors forcanine allergy. The canine allergy inhibitor of the present inventioncomprises a eukaryotic cell expression vector containing canineallergenic protein (such as Can f1 or Can f2) or a peptide or proteinthat comprises an antigenic epitope of such allergenic protein, incombination with a canine allergenic protein (such as Can f1 or Can f2)or a peptide or protein that comprises an antigenic epitope of suchallergenic protein.

In some embodiments, the present invention provides inhibitors for dustmite allergy. The dust mite allergy inhibitor of the present inventioncomprises a eukaryotic cell expression vector containing a dust miteallergy allergenic protein (such as Der P1 or Der F1) or a peptide orprotein that comprises an antigenic epitope of such allergenic protein,in combination with a mite allergy allergenic protein (such as Der P1 orDer F1) or a peptide or protein that comprises an antigenic epitope ofsuch allergenic protein.

In some embodiments, the present invention provides inhibitors forpeanut allergy. The peanut allergy inhibitor of the present inventioncomprises a eukaryotic cell expression vector containing a peanutallergy allergenic protein (such as Ara HII or Ara H5) or a peptide orprotein that comprises an antigenic epitope of such allergenic protein,in combination with a peanut allergy allergenic protein (such as Ara HIIor Ara H5) or a peptide or protein that comprises an antigenic epitopeof such allergenic protein.

In some embodiments, the present invention provides inhibitors forJapanese cedar allergy. The Japanese cedar allergy inhibitor of thepresent invention comprises a eukaryotic cell expression vectorcontaining a Japanese cedar allergy allergenic protein (such as Cry j1.1 or Cry j 1.2) or a peptide or protein that comprises an antigenicepitope of such allergenic protein, in combination with a Japanese cedarallergy allergenic protein (such as Cry j 1.1 or Cry j 1.2) or a peptideor protein that comprises an antigenic epitope of such allergenicprotein.

In some embodiments, the present invention provides inhibitors forblomia tropicalis allergy. The blomia tropicalis allergy inhibitor ofthe present invention comprises a eukaryotic cell expression vectorcontaining a blomia tropicalis allergy allergenic protein (such as Blot5) or a peptide or protein that comprises an antigenic epitope of suchallergenic protein, in combination with a blomia tropicalis allergyallergenic protein (such as Blo t5) or a peptide or protein thatcomprises an antigenic epitope of such allergenic protein.

The allergenic protein may be expressed in Escherichia coli oreukaryotic cells (for example, yeast or CHO cells), for example,molecular cloning methodology is used to incorporate the allergenicprotein coding sequence into the corresponding expression vector,causing the protein product to be expressed through the Escherichiacoli, yeast or CHO cell systems. Purification is then used to obtain theallergenic protein. Similarly, peptides or proteins may be designedwhich include antigenic epitopes of allergenic proteins. Nucleic acidsequences encoding such peptides or proteins can be incorporated intoexpression vectors and produced in host cells where they express thepeptide or protein which is then purified or peptides may besynthesized. Alternatively, the allergenic protein may be purified fromnatural sources.

The eukaryotic cell expression vector included in the compositions orkits of the invention may be an expression vector composed of a plasmidexpression vector, a viral expression vector or bacteriophage expressionvector. Plasmid DNA and chromosome DNA fragment-formed expression vectorand other expression vectors are well known and commonly used in thefield of genetic engineering. In some embodiments, the plasmid vectorpVAX1 (Invitrogen) is used. In some embodiments, the plasmid vectorprovax which has the CMV promoter, an hCG leader and bovine growthhormone poly A is used. In some embodiments, the plasmid vector is apcDNA3 plasmid (Invitrogen) which comprises a human cytomegalovirusimmediate-early (CMV) promoter, bovine growth hormone polyadenylationsignal (BGH polyA), T7 sequence, ColE1 origin of replication, and the JEvirus signal sequence.

In the eukaryotic cell expression vectors, the coding sequence for theallergenic protein or peptide or protein that comprises an antigenicepitope of such allergenic protein is operably linked to regulatorysequences required for eukaryotic expression. Examples of suitablepromoters include an RSV (Rous sarcoma virus) promoter, a CMV(cytomegalovirus) promoter such as the CMV immediate early promoter, anSV40 virus promoter, Mouse Mammary Tumor Virus (MMTV) promoter, HumanImmunodeficiency Virus (HIV) such as the HIV Long Terminal Repeat (LTR)promoter, Moloney virus, ALV, Epstein Barr Virus (EBV), as well aspromoters from human genes such as human Actin, human Myosin, humanHemoglobin, human muscle creatine and human metalothionein. Examples ofpolyadenylation signals useful to practice the present invention,include but are not limited to SV40 polyadenylation signals and LTRpolyadenylation signals. In addition to the regulatory elements requiredfor DNA expression, other elements may also be included in the DNAmolecule. Such additional elements include enhancers. The enhancer maybe selected from the group including but not limited to: human Actin,human Myosin, human Hemoglobin, human muscle creatine and viralenhancers such as those from CMV, RSV and EBV.

In some embodiments, the proportion of eukaryotic cell expression vectorto allergenic protein or peptide or protein that comprises an antigenicepitope of such allergenic protein is 1:5-5:1; the preferred option is:1:1 (the mole ratio is 1-20:100,000; the preferred molar ration is15:100,000).

In some embodiments, the inhibitor composition or combination of kitcomponents is introduced into the organism intramuscularly,intracutaneously/intradermally, transdermally, subcutaneously,intravenously and through mucosal tissue by means of injection,nebulizer/aerosol/spraying, nose drops, eye drops, orally, sublingual,buccal, vaginal, penetration, absorption, physical or chemical means; orit may be introduced into the organism through other physical mixture orpackage. The kit components do not have to be delivered together, nor dothey have to be delivered at the same site or by the same route ofadministration.

The pharmaceutical composition may be introduced by various meansincluding, for example, the needle injection, needleless injector, genegun, electroporation, and microprojectile bombardment.

The composition and kit components may be formulated by one havingordinary skill in the art with compositions selected depending upon thechosen mode of administration. Suitable pharmaceutical carriers aredescribed in the most recent edition of Remington's PharmaceuticalSciences, A. Osol, a standard reference text in this field.

For parenteral administration, formulations may be provided as asolution, suspension, emulsion or lyophilized powder in association witha pharmaceutically acceptable parenteral vehicle. Examples of suchvehicles are water, saline and dextrose solution. Liposomes andnonaqueous vehicles such as fixed oils may also be used. The vehicle orlyophilized powder may contain additives that maintain isotonicity(e.g., sodium chloride, mannitol) and chemical stability (e.g., buffersand preservatives). The formulation is sterilized by commonly usedtechniques. Injectable compositions may be sterile and pyrogen free.

The dosage administered varies depending upon factors such as:pharmacodynamic characteristics; its mode and route of administration;age, health, and weight of the recipient; nature and extent of symptoms;kind of concurrent treatment; and frequency of treatment. In someembodiments, the amount of composition used or the amount of combinationof kit components is generally 1250 μg/kg body weight/administration;with the composition or kit components administered once every 1-30days, preferably once every 7-14 days. In some embodiments, a singledose is administered. In some embodiments, multiple doses areadministered. In some embodiments, a total of 2-3 administrations areadministered.

In some embodiments, the compositions or kits are administered toindividuals who are suffering from an allergic reaction. In someembodiments, the compositions or kits are administered to individualswho are not suffering from an allergic reaction but who have beenexposed to the allergen or likely to have been exposed to the allergen.In some embodiments, the compositions or kits are administered toindividuals who are not suffering from an allergic reaction but who areknown to be allergic to the allergen, ie. who have previously hadallergic responses to the allergen.

The methods of the present invention are useful in the fields of bothhuman and veterinary medicine. Accordingly, the present inventionrelates to treatment and prevention of allergic reactions in mammals,birds and fish. The methods of the present invention can be particularlyuseful for mammalian species including human, bovine, ovine, porcine,equine, canine and feline species.

The Examples set out below include representative examples of aspects ofthe present invention. The Examples are not meant to limit the scope ofthe invention but rather serve exemplary purposes. In addition, variousaspects of the invention can be summarized by the following description.However, this description is not meant to limit the scope of theinvention but rather to highlight various aspects of the invention. Onehaving ordinary skill in the art can readily appreciate additionalaspects and embodiments of the invention.

EXAMPLES Example 1 Polypeptide Synthesis of Flea Salivary AllergenicPeptides and Construction of a Eukaryotic Cell Expression Vectors forExpression of the Same

The described flea salivary allergenic protein (FSA) possesses the aminoacid residue sequence as described in SEQ ID NO:2.

The synthesized polypeptide that comprises the amino acid residuesdescribed in SEQ ID NO:22 is named pep66. The synthesized polypeptidethat comprises amino acids described in SEQ ID NO:23 is named pep100.

Nucleotide sequences that encode pep66 have the nucleotide sequencedescribed in SEQ ID NO 24 and are named FAD66.

Nucleotide sequences that encode pep100 have the nucleotide sequencedescribed in SEQ ID NO 25 and are named FAD100.

Eukaryotic vectors that encode pep66 and pep100 comprise at least onenucleotide sequence described in SEQ ID NO 24 or SEQ ID NO: 25 and arenamed pcDF66 or pcDF100, respectively.

Flea Salivary Allergenic Protein was purchased from the Greer LaboratoryCompany (Lenoir, N.C., United States) and was formulated by thesystematic flea cultivation methods described by Lee, et al., inParasite Immunology 19:13-19, 1997. At the end of one adult year, femalefleas were obtained from isolated salivary glands of infected animals.Salivary gland cells were suspended in SDS-reduction buffer and agitatedin an oscillator for 30 seconds. Cells were pulverized and crude proteinwas stored at −20° C.

The described eukaryotic cell expression vectors comprising nucleotidesequences that encode FSA epitopes pep66 and pep100 are named pcDF66 orpcDF100, respectively.

1. Synthesis of FSA Polypeptides pep66, pep100, and Their Encoded Genes

We verified the amino acid sequences of MHC Class II epitopes of FSA andthe chemically synthesized peptides pep66 and pep100 using Epitlotsoftware. Sequences of the newly synthesized genes and protein productsare below:

Peptide 66-80: QEKEKCMKFCKKVCK (SEQ ID NO: 22), called pep66;

Peptide 100-114: GPDWKVSKECKDPNN (SEQ ID NO: 23), called pep100.

Nucleotide sequences that encode pep66 and pep100 have been named FAD66and FAD100, respectively. The nucleotide sequences comprise thefollowing sequences:

FAD66: CAAGAGAAAG AAAAATGTAT GAAATTTTGC (SEQ ID NO: 24 AAAAAAGTTT GCAAAFAD100: GGTCCTGATT GGAAAGTAAG CAAAGAATGC (SEQ ID NO: 25) AAAGATCCCAATAAC.2. Construction of FAD66 and FAD100 Expression Vectors Expression vectorpGFP (Clontech, Mountain View, Calif., U.S.A) was purchased as atemplate. We conducted Polymerase chain reaction (PCR) amplification ofFSA nucleotide sequences to label the FSA nucleotide sequence with theGreen Fluorescent Protein (GFP) gene. Primer extension was completed byuse of the P66F and PR primers as well as the P100F and PR primers.Sequences for the primers used in cloning method are as follows: P66F:5′-AAGCTTGCCA TGCAAGAGAA AGAAAAATGT ATGAAATTTT GCAAAAAAGT TTGCAAAGGTACCGCCATGG TGAGCAAGGG CGAGGA-3′ (SEQ ID NO:26) (the 13^(th) site-57^(th)site basic group from the 5′ terminal end of the amplification productsaid sequence is FAD66; the 58th site-63rd site basic group from the 5′terminal end of the amplification product is the Kpn I recognition site;the first through sixth basic nucleotide from the 5′ terminal end of theamplification product is the Hind III recognition site); PR: 5′-TTAGGTACCTTAC TTGTAC AGCTCGTCCAT-3′ (SEQ ID NO:27) (the 4^(th) site-9^(th)site basic group from the 5′ terminal end of the amplification productin said sequence is the Kpn I recognition site), P100F: 5′-AAGCTTGCCA TGGGTCCTGA TTGGAAAGTA AGCAAAGAAT GCAAAGATCC CAATAACGGT ACCGCCATGGTGAGCAAGGGCGAGGA-3′ (SEQ ID NO:28) (the 13^(th) site-57^(th) sitebasic group from the 5′ terminal end of the amplification product insaid sequence is FAD66; the 58^(th) site-63^(rd) site basic group fromthe 5′ terminal end of the amplification product is the Kpn Irecognition site; the 1^(st) site-6^(th) site basic group from the 5′terminal end of the amplification product in said sequence is the HindIII recognition site), PR: 5′-TTA GGTACCTTAC TTGTAC AGCTCGTCCAT-3′ (SEQID NO: 27) (the 4^(th) site-9^(th) site basic group from the 5′ terminalend of the amplification product is the Kpn I recognition site). PCRproduct and eukaryotic expression vector pcDNA3 (Invitrogen Corp.,Carlsbad, Calif., U.S.A.) were digested using BamH I and Hind III: Theamplification product was ligated into the plasmid using T₄ DNA ligase,and then transformed into Escherichia coli Top 10. After E. coli weregrown in incubators, plasmid DNA was extracted and restriction digestionwas performed using Kpn I to obtain a positive clones. Positive clonesincluded plasmid pcDF66-GFP containing FAD66 and GFP genes and plasmidpcDF100-GFP containing FAD100 and GFP genes. After using pcDF66-GFP andpcDF100-GFP for Kpn I digestion, low melting point agarose gelelectrophoresis was used to recover large fragments, and thenself-binding is conducted. Finally, the product is transformed intoEscherichia coli Top 10, the plasmid is extracted and restrictionendonuclease Kpn I digestion assay is used to obtain a positive clone.Obtained clones included FAD66 expression vector pcDF66 and FAD100expression vector pcDF100.

Normal simian kidney cells (CV1 cells) (purchased from the Institute ofCell Biology, Shanghai) were cultured in DMEM containing 10% fetal calfserum under 5% CO₂, and 37° C. Transfections of pcDF66-GFP andpcDF100-GFP were performed on the CV1 cells in a 35 mm culture dish with2.5×10⁵ cells per/ml and 2 ml per dish. Purification of the plasmids wasperformed in accordance with the methodology described in the Guidebookfor Molecular Cloning Experimentation (3^(rd) edition) (Chinesetranslation) (translated by Huang Peitang et al., Science PublishingCompany, published September 2002). A positive ion liposome medium(Lipofectamine™2000, Invitrogen) was used to transfect and culture theCV1 cells according to the manufacturer's instructions (Invitrogen,Calif., USA). After 24 hours of incubation, the cell culture is observedunder fluorescence microscopy showing pcDF100-GFP and pcDF66-GFP wasexpressed in eukaryotic cells. The results demonstrate that pcDF100-GFPand pcDF66-GFP also may be expressed in eukaryotic cells.

Example 2 Flea Allergy Dermatitis Inhibitor as Therapy for Flea AllergyDermatitis

Experiments in which Kunming white, BALB/c and C57BL/6 mice areimmunized with a vector comprising FSA protein and nucleotide sequencesthat encode FSA proteins, or proteins thereof, demonstrate thatimmunization is an effective therapy for flea allergy dermatitis. Usefulvectors for immunization can comprise a eukaryotic cell expressionvector further comprising a nucleotide sequences encoding FSA protein(for example, pcDF66 or pcDF100), and either FSA synthesized peptides(pep66 or pep100) or FSA protein (flea salivary allergenic protein). Theimmunization efficacy is superior to that of an immunization vectormerely comprising a eukaryotic cell expression vector that includednucleotide sequences that encodes either FSA peptides or FSA protein.Inhibition studies demonstrate that the DNA sequences encoding differentepitopes have different efficacies. For instance, FAD100 appears to havea stronger therapeutic effect in suppressing Flea Allergy Dermatitis.Results in each strain of mouse were similar, indicating thatimmunosuppressive activity of the immunization is not limited to MHCgenetic backgrounds. Therefore, we can directly deduce from the aboveresults that through use of a FAD Inhibitor comprising a eukaryotic cellexpression vector which further comprises a nucleotide sequence thatencodes FSA protein and either a FSA protein or a FSA peptide, it ispossible to effectively inhibit flea allergy dermatitis.

T-cell proliferation experiments and related cytokine expansionexperiments demonstrate that a FAD Inhibitor, comprising a eukaryoticcell expression vector that encodes FSA protein and an FSA protein orFSA peptide, inhibit antigen-specific T-cell proliferation therebysuppressing an allergic reaction. Immunosuppression may be inducedthrough IL-10, thus inhibiting IL-5, IL-13 and other cytokine expressionlevels. The FAD Inhibitor in the present invention may effectivelyprevent and/or treat flea allergy dermatitis, especially those casescaused by Ctenocephalides felis.

1. Kunming White Mice Experiments.

Three Hundred Sixty (360) female Kunming white mice were divided into atotal of 12 groups of equal numbers. Each mouse in the first group wasimmunized with 100 microliters of 0.9% NaCl aqueous solution containing100 micrograms of pcDF66. Each mouse in the second group was immunizedwith 100 microliters of 0.9% NaCl aqueous solution containing 100micrograms pep66. Each mouse in the third group was immunized with 100microliters of 0.9% NaCl aqueous solution containing 50 micrograms ofpcDF66 and 50 micrograms of pep66. Each mouse in the fourth group wasimmunized with 100 microliters of 0.9% NaCl aqueous solution containing100 micrograms of pcDF100. Each mouse in the fifth group was immunizedwith 100 microliters of 0.9% NaCl aqueous solution containing 100micrograms of pep100. Each mouse in the sixth group was immunized with100 microliters of 0.9% NaCl aqueous solution containing 50 microgramsof pcDF100 and 50 micrograms of pep100. Each mouse in the seventh groupwas immunized with 100 microliters of 0.9% NaCl aqueous solutioncontaining 50 micrograms of pcDF66 and 50 micrograms of pep100. Eachmouse in the eighth group was immunized with 100 microliters of 0.9%NaCl aqueous solution containing 50 micrograms of pcDF100 and 50micrograms of pep66. Each mouse in the ninth group was immunized with100 microliters of 0.9% NaCl aqueous solution containing 100 microgramsof inactivated flea antigen (purchased from the Greer Lab Company, N.C.,United States). Each mouse in the tenth group was immunized with 100microliters of 0.9% NaCl aqueous solution containing 50 micrograms ofinactivated flea antigen (purchased from the Greer Lab Company, N.C.,United States) and 50 micrograms of pcDF66. Each mouse in the eleventhgroup was immunized with 100 microliters of 0.9% NaCl aqueous solutioncontaining 50 micrograms of inactivated flea antigen (purchased from theGreer Lab Company, N.C., United States) and 50 micrograms of pcDF100.Each mouse in the twelfth group was immunized with 100 microliters of0.9% NaCl aqueous solution containing 100 micrograms of pcDNA3 to serveas a control. Fourteen days after the first immunization, a boosterimmunization was administered in the same dosage amount. Seven daysafter the booster immunization, skin tests were conducted using thefollowing methods.

Hair was removed from the ventral murine chest cavity and anintracutaneous injection of 30 μl of inoculation of 1 μg/ul FSA proteinwas injected into 10 subjects. At the same time a histamine solution(with a concentration of 0.01% histamine) and PBS was injected in equalamounts to serve as positive controls and negative controls. There were10 subjects for each control group. Twenty minutes after each injection,we measured blister diameters in micrometers. The t test resultsindicated that pcDF100 and pep100 compound immunization (the sixthgroup) demonstrated a notable difference (P<0.05) when compared topcDF100 single immunization (the fourth group) or pep100 singleimmunization (the fifth group). There was a notable difference (P<0.05)between pcDF66 and pep66 compound immunization (the third group) andpcDF66 single immunization (the first group), while there was no notabledifference with pep66 single immunization (the second group). Fleaantigen and pcDF66 (the tenth group) or pcDF100 (the eleventh group)compound immunizations were notably different (P<0.05) than the resultsgenerated by flea antigen alone (the ninth group), The tenth andeleventh groups did not display any notable difference as compared topcDF66 (the first group) or pcDF100 (the fourth group) immunizations.There was no difference in blister diameters measured in the sixth andseventh groups as compared to the expression vector or epitopepolypeptide single immunity groups (Groups 1, 2, 3 and 4).

Based on the preceding skin test results, we can conclude that aeukaryotic cell expression vector comprising FSA protein or a nucleotidesequence encoding FSA peptide and said FSA peptide, or a eukaryotic cellexpression vector comprising FSA protein or a nucleotide sequence thatencodes FSA peptides and FSA protein reduces skin allergies in anantigen-specific way. These results indicate that reduction of allergicreactions on the skin may be induced through immunosuppression.

2. BALB/c and C57B/6 Mice Skin Test Results

In order to further verify the results obtained above with Kunming whitemice, two pure strains of mice (BALB/c and C57B/6) were tested todetermine whether immunosuppression of FAD was MHC restricted.Experimental methodologies were the same as those methodologiesperformed in the Kunming white mice. The t test results indicate thatpcDF100 and pep100 immunization (the sixth group) had no notabledifference (P<0.05) as compared to pcDF100 single immunization (thefourth group) or pep100 single immunization (the fifth group). Resultsfrom the flea antigen and pcDF100 compound immunization (the eleventhgroup) demonstrated a very significant difference (P<0.01) compared tothe results generated by immunizations of flea antigen (the ninth group)or pcDF100 (the fourth group). There was no significant difference amongimmunizations of Flea antigen and pcDF66 immunization (the tenth group),flea antigen immunization alone (the ninth group), or pcDF66immunization alone (the first group). There was no significantdifference among pcDF100 and pep66 immunization (the eighth group),pcDF100 immunization alone (the fourth group), or pep66 immunizationalone (the second group).

The preceding results indicate that anti-allergic immunosuppression isantigen-specific. For example, immunization with pcDF100 and pep100 wasmore effective in as compared to immunization with pcDF100 or pep100alone. Immunization with the flea antigen and pcDF100 were moreeffective than any immunization vectors that included only onecomponent.

Differences also exist in the level of immunosuppression among thosegroups that effectively treated FAD. For example, the flea antigen andpcDF100 compound immunity group had more of an effectiveimmunosuppressive effect than compared any of the single immunitygroups. The t test results indicate that pcDF100 and pep100 compoundimmunity (the sixth group) are significantly different (P<0.05) whencompared to results of immunization using pcDF100 alone (the fourthgroup) or pep100 alone (the fifth group). There are significantdifferences (P<0.05) in the immunosuppressive effect of theimmunizations performed with pcDF100 and pep66 compound immunity (theeighth group) as compared to pcDF100 alone (the fourth group) or pep66alone (the second group). There was an extremely significant difference(P<0.01) in immunizations using Flea antigen and pcDF100 (the eleventhgroup) as compared to flea antigen (the ninth group) or pcDF100 alone(the fourth group).

We interpret our results to conclude that anti-allergicimmunosuppression is antigen-specific. For example, immunization wasmore effective in the pcDF100 and pep100 compound immunity group ascompared to the pcDF100 single immunity group or the pep100 singleimmunity group. In addition, pep66 and pep100 peptides may havecross-reactivity. For example, immunization was more effective withpcDF100 and pep66 immunization as compared to pcDF100 single immunitygroup or the pep66 single immunity group. Immunization using the fleaantigen and pcDF66 compound immunity did not produce clearimmunosuppression. These results are consistent with those obtained inthe BALB/c group. Although there are slight differences in theeffectiveness of the immunization among each strain of mice studied,experimental results of the three different strains of mice sameconclusion: immunization vectors comprising eukaryotic cell expressionvectors further comprising FSA protein or nucleotide sequences thatencode FSA peptides and FSA peptides; or eukaryotic cell expressionvectors comprising FSA protein or nucleotide sequences that encode FSApeptides and FSA protein may mount effective anti-allergicimmunosuppression.

Example 3 T-Cell Proliferation in Immunized Mice

Three Hundred Sixty (360) BALB/c mice and three hundred sixty C57B/6mice were each divided into 12 groups of 30 mice per group. Immunizationwas performed in accordance with the methodology described in Example 2.At seven days after the booster immunization, splenic T-cells wereisolated and T-cell expansion activity was tested. The specificmethodology was: under aseptic conditions, splenic cells were taken frommice and used to form a single-cell suspension fluid. A hemolyticsolution was used to remove red blood cells, which were then washedthree times using PBS fluid. The cells were centrifuged and a cell counttaken. Cell concentration was adjusted to 1×10⁶ cells/ml, and each cellsuspension from each animal was divided into four experimental groupsand plated into a 96-well culture plate. To one group, 100 μl Con-A(mitogen) was added to a final concentration of 5 μg/ml. Specificantigen (flea antigen) was added to serve as a stimulant to a finalconcentration of 5 μg/ml to the second group. No stimulant was added fora negative control group, and 100 μl BSA was added to a finalconcentration of 2 μg/ml to serve as another unrelated antigen. Afterbeing incubated at 37° C. in a CO₂ culture for 48 h, 100 μl MTS wasadded to each well at a final concentration of 5 mg/ml. After 4 hours ofincubation, an enzyme labeler was used to read the OD value at 492 nmand calculate the stimulation index (SI=tested OD÷non-stimulated OD).T-cell proliferation results for the BALB/c mice indicated thateukaryotic cell expression vectors comprising a nucleotide sequence thatencodes FSA peptide flea salivary and said FSA peptide generate notableantigen-specific immunosuppression of FAD. For example, immunization wasmore effective from the vectors comprising pcDF100 and pep100 (the sixthgroup) and the vectors comprising flea antigen and pcDF100 (the eleventhgroup) as compared to immunization with vectors comprising singlecomponents. In addition, there was no clear immunosuppressiondemonstrated by immunization vectors comprising the pcDF66 and pep66group (the third group), the flea antigen and pcDF66 group (the tenthgroup) as compared to the corresponding single immunity groups. Resultsusing vectors comprising the pcDF100 and pep100 compound immunity groupand the flea antigen and pcDF100 compound immunity group are consistentwith the immunosuppression effect shown by the skin tests. There was anextremely significant difference (P<0.01) in the immunosuppressiveeffect of the immunization vectors comprising the pcDF100 and pep66 (theeighth group) as compared to the immunosuppressive effect seen in thecorresponding single immunity group.

C57B/6 T-cell proliferation results indicate that eukaryotic cellexpression vectors containing nucleotide sequences that encode FSApeptide and said FSA peptide may produce clear antigen-specificimmunosuppression. For example, there is a clear difference (P<0.05) inthe effect of immunization of pcDF66 and pep66 compound immunity (thethird group) as compared to the corresponding single immunity groups(the first group and the second group). Additionally, there was anextremely significant difference (P<0.01) in the effect of immunizationin the pcDF100 and pep100 compound immunity group (the sixth group) ascompared to the corresponding single immunity groups (the fourth groupand the fifth group). The immunization of mice using vector comprisingthe flea antigen and pcDF66 (the tenth group) is clearly more effective(P<0.05) than the corresponding single immunity groups (the ninth groupand the first group). There was an extremely significant difference(P<0.01) in the effect of the immunization of the flea antigen andpcDF100 compound immunity group (the eleventh group) as compared to thecorresponding single immunity groups (the ninth group and the fourthgroup). In addition, cross-reactivity exists between the two epitopes.For example, there is a clear difference (P<0.05) in T-cellproliferation profiles with the pcDF66 and pep100 compound immunitygroup (the seventh group) and the corresponding single immunity groups(the first group and the fifth group). There is also a clear difference(P<0.05) in T-cell proliferation profiles of the pcDF100 and pep66compound immunity group (the eighth group) and the corresponding singleimmunity groups (the fourth group and the second group). The T-cellproliferation profiles of in the pcDF100 and pep100 compound immunitygroup, the pcDF100 and pep66 compound immunity group and the fleaantigen and pcDF100 compound immunity group results were all consistentwith the skin test results.

Example 4 Changes in the Cytokine Levels of Immunized Mice

There were 360 Kunming white mice, 360 BALB/c mice, and 360 C57B/6 mice,and each set of subjects strain was divided into 12 groups of 30 mice.Immunization was performed in accordance with the method described inExample 2. Seven days after the booster immunization, the spleen wasexcised and total RNA (TRIZOL, Dingguo Biological Company) isolated.Reverse transcription for cDNA was performed in accordance with theDalianbao Biological Company's RNA RT-PCR operating handbook. Briefly 1μg of purified total RNA was placed in a 250 μL centrifuge tube. Thenthe following reagents were added in sequence: 4 μl MgCl₂, 2 μl 10×buffer solution, 8.5 μl DEPC water, 2 μl dNTP mixture, 0.5 μl RNaseinhibitor, 0.5 μl M-MLV reverse transcriptase (Promega), 0.5 μL Oligo(dT)₁₂ primer. The response conditions were 42° C. for 30 min, 99° C.for 5 min and 5° C. for 5 minutes. The gene family hypoxanthinephosphoribosyltransferase (HPRT) was used as the internal sourceexpression standard. The various groups' cDNA concentrations wereadjusted to make all sample concentrations consistent. Then 2 μl of cDNAwere used to conduct PCR amplification assessing the expression levelsof the three cytokine genes: IFN-γ, IL-4, and IL-10. The reaction'srequired primer and PCR reaction conditions are as indicated in Table 1.(Because the gene family HPRT has fixed expression in vivo, it is usedas the template for the control's internal source expression standard).

TABLE 1 HPRT, IFN-γ, IL-4 and IL-10 Primer Sequence and PCR ReactionSpecifications. Target gene Primer Response conditions HPRT 5′GTTGGATACAGGCCAGACTTTGTTG (SEQ ID NO: 29) 94° C. 30 sec, 60° C. 30 sec3′ GAGGGTAGGCTGGCCTATGGCT (SEQ ID NO: 30) and 72° C. 40 sec IFN-γ 5′CATTGAAAGCCTAGAAAGTCTG (SEQ ID NO: 31) 94° C. 30 sec, 58° C. 30 sec 3′CTCATGGAATGCATCCTTTTTCG (SEQ ID NO: 32) and 72° C. 40 sec IL-4 5′GAAAGAGACCTTGACACAGCTG (SEQ ID NO: 33) 94° C. 30 sec, 54° C. 30 sec 3′GAACTCTTGCAGGTAATCCAGG (SEQ ID NO: 34) and 72° C. 40 sec IL-10 5′CCAGTTTACCTGGTAGAAGTGATG (SEQ ID NO: 35) 94° C. 30 sec, 56° C. 30 sec 3′TGTCTAGGTCCTGGAGTCCAGCAGACTCAA (SEQ ID NO: 36) and 72° C. 40 sec

Bio-Rad Image software (Quantity One 4.2.0) was used to analyze imagestaken of the electrophoresis gels of the PCR products. Expressionprofile results obtained were generally consistent each strain of mouse.IL-10 expression levels in the pcDF66 and pep66 compound immunity (Group3) were higher than pcDF66 single immunity (Group 1) and the pep66single immunity (Group 2). IL-10 expression levels in the pcDF100 andpep100 compound immunity (Group 6) were higher than pcDF100 singleimmunity group (Group 4) or pep100 single immunity group (Group 5).IL-10 expression levels in the flea antigen and pcDF66 compound immunitygroup (Group 10) were higher than flea antigen (Group 9) or pcDF66single immunity groups (Group 1). IL-10 expression levels in the fleaantigen and pcDF66 compound immunity group (Group 11) were higher thanflea antigen (Group 9) or pcDF100 single immunity groups (Group 4).There was no clear difference in the IL-4 and IFN-γ expression levelsamong the various groups. The results suggest that immunizationconducted with eukaryotic cell expression vector comprising nucleotidesequences that encode FSA and said FSA protein or said FSA peptidecompound enhanced IL-10 expression levels.

In addition, expression profiles were generated on IL-5 and IL-13 in thethree types of mice. Results indicated that for the eukaryotic cellexpression vectors comprising nucleotide sequences that encode FSApeptides and said FSA protein or peptide (Group 3, Group 6, Group 10 andGroup 11), IL-5 and IL-13 levels were clearly lower than those of therespective single immunity groups.

Example 5 Detection of Blood IgE Levels in Immunized Mice

Two groups of 360 BALB/c and 360 C57B/6 mice were each divided into 12groups of 30 mice each and immunization was performed as described inExample 2. Blood was taken intravenously from the eye socket prior toimmunization and 14 days after the booster immunization. The blood wasseparated by centrifugation and IgE levels were assessed using ELISA.The coated antigen used on the ELISA plates was flea antigen purchasedfrom Greer Laboratory (Lenoir, N.C., United States). The first componenton the ELISA plate was separated blood sera and the second componentused for binding was the sheep anti-mice IgE antibody labeled withhorseradish peroxidase antioxidant enzyme. The substrate was added tothe system after antibody was bound to the antigens and the enzymelabeler was used to read the OD values at 492 nm. The IgE production inKunming white mice, BALB/c mice, C57B/6 mice after immunization aregenerally consistent for all three groups of mice. Except for the fleaantigen single immunity group (the ninth group), IgE levels for thegroups were relatively low. The IgE levels produced after immunizationwith the flea antigen single immunity group (the ninth group) were thehighest levels across all sets of mice. IgE levels were greatly reducedin the group immunized with flea antigen and pcDF66 (the tenth group)and the group immunized with the flea antigen and pcDF100 (the 11 group)as compared to the flea antigen single immunity group (the ninth group).This indicates that a eukaryotic cell expression vector comprisingnucleotide sequences that encode FSA peptides and said FSA proteinreduces IgE levels after immunization.

Example 6 Feline Allergenic Protein Antigen Fe1 d. I.1 (Fe1 d I With theMinor B Leader) Encoded Genetic Clone and Eukaryotic Expression Assay

1. The following sequences were used as Fe1 d I.1 primers. Primer wereartificially synthesized:

(a) Fe1 d I.1 P1 5′ primer: AAGCTTGGATGTTAGACGC (SEQ ID NO 37) (b) Fe1 dI.1 P2 3′ primer: GGTACCTTAACACAGAGGAC (SEQ ID NO 38)

2. Fe1 d I.1 expression vector construction

Fe1 d I.1 cDNA was used as a template for PCR amplification of the Fe1 dI.1 gene using Fe1 d I.1 P1 and Fe1 d I.1 P2. The primers are furtherdescribed below:

(a) Fe1 d I.1 P1 5′-AAGCTTGGATGTTAGACGC-3′ (SEQ ID NO 37) (the 1^(st)site-6^(th) site basic group from the 5′ terminal end of the primer inthis sequence is the Hind III recognition site, the 9^(th) site-11^(th)site is the original initiator code);

(b) Fe1 d I.1 P2 5′-GGTACCTTAACACAGAGGAC-3′ (SEQ ID NO 38) (the 1^(st)site-6^(th) site basic group from the 5′ terminal end of the primer inthis sequence is the Kpn I recognition site; the 7^(th) site-9^(th) siteis the original termination code).

Kpn I and Hind III, respectively, were used for digesting the PCRproduct and eukaryotic expression vector pVAX1 (Invitrogen Corp.).Digested nucleotide fragments were then ligated using T₄ DNA ligase. Theresultant plasmid product was transformed into Escherichia coli Top 10,grown for maximum copy number, and then the plasmid isolated usingmethods known by those of ordinary skill in the art. We performed arestriction digestion using the Kpn I and Hind III endonucleases.Selected plasmid clones named pVAX1-Fe1 d I.1 comprised the pVaxplasmids sequence and the Fe1 d I.1 gene. Through sequential analysis(Augct Co. Ltd., Beijing China), further analysis was performed toobtain the final Fe1 d I.1 P1 expression vector, pFe1d I.1.

3. pFe1dI.1 Eukaryotic Expression

Normal simian kidney cells (CV1 cells, purchased from the Shanghai CellInstitute) were cultured in DMEM containing 10% fetal calf serum under5% CO₂ and 37° C. conditions. 2.5×10⁵ cells per/ml were pipetted in a 35mm culture dishes in a 2 mL volume. Transfections were performed usingstandard methods. Briefly, purification of the plasmids was performed inaccordance with the methodology in the Guidebook for Molecular CloningExperimentation (third edition, Chinese translation) (translated byHuang Peitang et al., Science Publishing Company, published September2002). A positive ion liposome medium (Lipofectamine™ 2000, Invitrogen)was used to transfect the cultured CV1 cells according to themanufacturer's instructions (Invitrogen, Calif., USA). After 24 hours oftransfection, the cells were collected and an RNA extraction reagent(TRIZOL, Dingguo Biological Company) was used to isolate total cellularRNA. In order to prevent contamination, total extracted RNA wasseparately loaded into several EP tubes. A micropipette was used tocarefully suction 2 ul of total RNA extracted and RT-PCR reagent used toexpand total cellular cDNA. After the specimen was added, reversetranscription was performed at 42° C. for 30-60 minutes, denaturation at99° C. for 5 minutes. and The reaction tube was set aside at 5° C. for 5minutes prior to extraction for use. Using the isolated cDNA as atemplate for gene amplification PCR was performed using Fe1dI.1P1 andFe1dI.1P2 as the primers. The PCR products were subject to low meltingpoint agarose gel analysis to detect Fe1dI.1 positive bands. The resultsdemonstrate that Fe1 dI.1 is expressed in eukaryotic cells.

Example 7 Feline Allergenic Protein Antigen Fe1 d. I.2 Genetic Clone andEukaryotic Expression Assay

1. The Following Sequences Were Used as Fe1 d I.2 Primers. Primers WereArtificially Synthesized:

-   -   (a) Fe1 d I.2 P1 5′ primer: AAGCTTGGATGAAGGGGGCTC (SEQ ID NO:39)    -   (b) Fe1 d I.2 P2 3′ primer: GGTACCTTAACACAGAGGAC (SEQ ID NO:40)        2. Fe 1 d I.2 Expression Vector Construction

Fe1 d I.2 cDNA was used as a template for PCR amplification using Fe1 dI.2P1 and Fe1 d I.2P2 primers to generate copies of the Fe1 d I.2 gene.The primers are further described below: (a) Fe1 d I.2P15′-AAGCTTGGATGAAGGGGGCTC-3′ (SEQ ID NO:39) (the 1^(st) site-6^(th) sitebasic group from the 5′ terminal end of the primer is the Hind IIIrecognition site; the 9^(th) site-11^(th) site from the terminal end ofthe primer is the original initiator code); (b) Fe1 d I.2 P25′-GGTACCTTAACACAGAGGAC-3′ (SEQ ID NO:40) (the 1^(st) site-6^(th) sitebasic group from the 5′ terminal end of the primer is the Kpn Irecognition site, the 7^(th) site-9^(th) site from the terminal end ofthe primer is the original termination code).

Kpn I and Hind III in succession were used for digesting the PCR productand eukaryotic expression vector, pVAX1 (Invitrogen Corp.). The digestedplasmid and PCR product were ligated using T₄ DNA ligase. The productwas transformed into Escherichia coli Top 10, the plasmid extracted, andrestriction endonuclease Kpn I and Hind III digestion assay used toobtain a positive clone, that is, plasmid pVAX1-Fe1 d I.2 containing theCan f 1 gene. Through sequential analysis (Augct Co. Ltd., BeijingChina), further assay correction was performed to obtain the Fe1 d I.2expression vector, pFe1d I.2.

3. pFe1dI.2 Eukaryotic Expression

The pFe1 d I.2 expression vector was subjected to the digestion,transformation, isolation, and ligation protocols previously describedin Example 6, Section 3. Isolated PCR products were subjected to lowmelting point agarose gel testing to confirm Fe1dI.2 positive bands. Theresults prove that Fe1dI.2 is expressed in eukaryotic cells.

Example 8 Canine Allergenic Protein Antigen Can f 1 Genetic Clone andEukaryotic Expression Assay

1. Can f 1 Primer Synthesis

The following sequences were used as Can f 1 primers. Primers wereartificially synthesized:

Can f 1 P1 5′ primer: AAGCTTATGAAGACCCTGCTCCTCAC (SEQ ID NO: 41) Can f 1P2 3′ primer: GGTACCCTACTGTCCTCCTGGAGAGC (SEQ ID NO: 42)2. Can f 1 Expression Vector Construction

Can f 1 cDNA was used as a template to perform PCR amplification usingCan f 1 P1 and Can f 1 P2 primers to generate copies of the Can f 1gene. The primers are further described below: (a) Can f 1 P15′-AAGCTTATGAAGACCCTGCTCCTCAC-3′ (SEQ ID NO:41) (the 1^(st) site-6^(th)site basic group from the 5′ terminal end of the primer sequence is theHind III recognition site; the 7^(th) site-9^(th) site from the terminalend of the primer sequence is the original initiator code); (b) Can f 1P2 5′-GGTACCCTACTGTCCTCCTGGAGAGC-3′ (SEQ ID NO:42) (the 1^(st)site-6^(th) site basic group from the 5′ terminal end of the primersequence is the Kpn I recognition site; the 7^(th) site-9^(th) site fromthe %′ terminal end of the sequence is the original termination code).

PCR fragments and pVAX1 (Invitrogen, Inc.) were subjected to thedigestion, transformation, isolation, and ligation methods as describedin Example 7, Section 2. Positive clones were selected and analyzedusing sequential analysis discussed in Example 7, Section 2 to obtainthe Can f 1 expression vector, pCanf1.

3. pCanf1 Eukaryotic Expression

The pCanf1 expression vector was subjected to transfection, total RNAisolation, and RT-PCR protocols as previously described in Example 6,Section 3. Isolated PCR products were subjected to low melting pointagarose gel testing to confirm pCanf1 positive bands. The results provethat pCanf1 is expressed in eukaryotic cells.

Example 9 Canine Allergenic Protein Antigen Can f 2 Genetic Clone andEukaryotic Expression Assay

1. Can f 2 Primer Synthesis

The following sequences were used as Can f 2 primers. Primers wereartificially synthesized:

Can f 2 P1 5′ primer: AAGCTT ATGCAGCTCCTACTGCTG (SEQ ID NO: 43) Can f 2P2 3′ primer: GGTACCCTAGTCTCTGGAACCC (SEQ ID NO: 44)2. Can f 2 Expression Vector Construction

Can f 2 CDNA cDNA was used as a template to perform PCR amplificationusing Can f 2 P1 and Can f 2 P2 primers to generate copies of the Can f2 gene. The primers are further described below: (a) Can f 2 P15′-AAGCTT ATGCAGCTCCTACTGCTG-3′ (SEQ ID NO:43) (the 1^(st) site-6^(th)site basic group from the 5′ terminal end of the primer sequence is theHind III recognition site; the 7^(th) site-9^(th) site is the originalinitiator code); (b) Can f 2 P2 5′ GGTACCCTAGTCTCTGGAACCC-3′ (SEQ IDNO:44) (the 1^(st) site-6^(th) site basic group from the 5′ terminal endof this primer sequence is the Kpn I recognition site, the 7^(th)site-9^(th) site is the original termination code).

PCR fragments and pVAX1 (Invitrogen, Inc.) were subjected to thedigestion, transformation, isolation, and ligation methods as describedin Example 7, Section 2. Positive clones were selected and analyzedusing sequential analysis discussed in Example 7, Section 2 to obtainthe Can f 2 expression vector, pCanf2.

3. pCanf2 Eukaryotic Expression

The pCanf2 expression vector was subjected to transfection, total RNAisolation, and RT-PCR protocols as previously described in Example 6,Section 3. Isolated PCR products were subjected to low melting pointagarose gel testing to confirm pCanf2 positive bands. The results provethat pCanf2 is expressed in eukaryotic cells.

Example 10 Dust Mite Allergenic Protein Antigen Der p 1 Genetic Cloneand Eukaryotic Expression Assay

1. Der p 1 Primer Synthesis

The following sequences were used as Der p 1 primers. Primers wereartificially synthesized:

Der p 1 P1 5′ primer: AAGCTTAACATGAAAATTGTTTTGG (SEQ ID NO: 45) Der p 1P2 3′ primer: GGTACCGTTTAGAGAATGACAACAT (SEQ ID NO: 46)2. Der p 1 Expression Vector Construction

Der p 1 cDNA was used as a template to perform PCR amplification usingDer p 1 P1 and Der p 1 P2 primers to generate copies of the Der p 1gene. The primers are further described below:

(a) Der p 1 P1 5′-AAGCTTAACATGAAAATTGGTTTTGG-3′ (SEQ ID NO:45) (the1^(st) site-6^(th) site basic group from the 5′ terminal end of theprimer sequence is the Hind III recognition site, the 10^(th)site-12^(th) site is the original initiator code);

(b) Der p 1 P2 5′-GGTACCGTTTAGAGAATGACAACAT-3′ (SEQ ID NO:46) (the1^(st) site-6^(th) site basic group from the 5′ terminal end of theprimer sequence is the Kpn I recognition site, the 9^(th) site-11^(th)site is the original termination code).

PCR fragments and pVAX1 (Invitrogen, Inc.) were subjected to thedigestion, transformation, isolation, and ligation methods as describedin Example 7, Section 2. Positive clones were selected and analyzedusing sequential analysis discussed in Example 7, Section 2 to obtainthe Der p 1 expression vector, pDerp1.

3. Der p 1 Eukaryotic Expression

The pDerp1 expression vector was subjected to transfection, total RNAisolation, and RT-PCR protocols as previously described in Example 6,Section 3. Isolated PCR products were subjected to low melting pointagarose gel testing to confirm pDerp1 positive bands. The results provethat pDerp1 is expressed in eukaryotic cells.

Example 11 Peanut Allergenic Protein Antigen Ara h II Genetic Clone andEukaryotic Expression Assay

1. Ara h II Primer Synthesis

The following sequences were used as Ara h II primers. Primers wereartificially synthesized:

Ara h II P1 5′ primer: AAGCTTCTCATGCAGAAGAT (SEQ ID NO: 47) Ara h II P23′ primer: GGTACCTTAGTAT CTGTCTC (SEQ ID NO: 48)2. Ara h II Expression Vector Construction

Ara h II cDNA was used as a template to perform PCR amplification usingAra h II P1 and Ara h II P2 primers to generate copies of the Ara h IIgene. The primers are further described below: (a) Ara h II P15′-AAGCTTCTCATGCAGAAGAT-3′ (SEQ ID NO:47) (the 1^(st) site-6^(th) sitebasic group from the 5′ terminal end of the primer sequence is the HindIII recognition site, the 10^(th) site-12^(th) site is the originalinitiator code), Ara h II P2 5′-GGTACCTTAGTATCTGTCTC-3′ (SEQ ID NO:48)(the 10^(th) site-6^(th) site basic group from the 5′ terminal end ofthe primer sequence is the Kpn I recognition site, the 7^(th)site-9^(th) site from the 5′ terminal end of the primer sequence is theoriginal termination code).

PCR fragments and pVAX1 (Invitrogen, Inc.) were subjected to thedigestion, transformation, isolation, and ligation methods as describedin Example 7, Section 2. Positive clones were selected and analyzedusing sequential analysis discussed in Example 7, Section 2 to obtainthe Ara h II expression vector, pArahII.

3. Ara h II Eukaryotic Expression

The pArahII expression vector was subjected to transfection, total RNAisolation, and RT-PCR protocols as previously described in Example 6,Section 3. Isolated PCR products were subjected to low melting pointagarose gel testing to confirm pArahII positive bands. The results provethat pArahII is expressed in eukaryotic cells.

Example 12 Peanut Allergenic Protein Antigen Ara h 5 Genetic Clone andEukaryotic Expression Assay

1. Ara h 5 Primer Synthesis

The following sequences were used as Ara h 5 primers. Primers wereartificially synthesized:

Ara h 5 P1 5′ primer: AAGCTTATGTCGTGGCAAAC (SEQ ID NO: 49) Ara h 5 P2 3′primer: GGTACCTAAAGACCCGTATC (SEQ ID NO: 50)2. Ara h 5 Expression Vector Construction

Ara h 5 cDNA was used as a template to perform PCR amplification usingAra 5 P1 and Ara h 5 P2 primers to generate copies of the Ara h 5 gene.The primers are further described below: (a) Ara h 5 P15′-AAGCTTATGTCGTGGCAAAC-3′ (SEQ ID NO:49) (the 1^(st) site-6^(th) sitebasic group from the 5′ terminal end of the primer sequence is the HindIII recognition site; the 7^(th) site-9^(th) site is the originalinitiator code); (b) Ara h 5 P2 5′-GGTACCTAAAGACCCGTATC-3′ (SEQ IDNO:50) (the 1^(st) site-6^(th) site basic group from the 5′ terminal endof the primer sequence is the Kpn I recognition site; the 7^(th)site-9^(th) site from the 5′ terminal end of the primer sequence is theoriginal termination code).

PCR fragments and pVAX1 (Invitrogen, Inc.) were subjected to thedigestion, transformation, isolation, and ligation methods as describedin Example 7, Section 2. Positive clones were selected and analyzedusing sequential analysis discussed in Example 7, Section 2 to obtainthe Ara h 5 expression vector, pArah5.

3. pArah5 Eukaryotic Expression

The pArah5 expression vector was subjected to transfection, total RNAisolation, and RT-PCR protocols as previously described in Example 6,Section 3. Isolated PCR products were subjected to low melting pointagarose gel testing to confirm pArah5 positive bands. The results provethat pArah5 is expressed in eukaryotic cells.

Example 13 Japanese Cedar (Cryptomeria japonica) Pollen AllergenicProtein Antigen Cry j 1.1 Genetic Clone and Eukaryotic Expression Assay

1. Cry j 1.1 Primer Synthesis

The following sequences were used as Cry j 1.1 primers. Primers wereartificially synthesized

Cry j 1.1 P1 5′ primer: AAGCTTATGGATTCCCCTTGCTTAT (SEQ ID NO: 51) Cry j1.1 P2 3′ primer: GGTACCATCAACAACGTTTAGAG (SEQ ID NO: 52)2. Cry j 1.1 Expression Vector Construction

Cry j 1.1 cDNA was used as a template to perform PCR amplification usingCry j 1.1 P1 and Cry j 1.1 P2 primers to generate copies of the Cry j1.1 gene. The primers are further described below:

(a) Cry j 1.1 P1 5′-AAGCTTATGGATTCCCCTTGCTTAT-3′ (SEQ ID NO:51) (the1^(st) site-6^(th) site basic group from the 5′ terminal end of theprimer sequence is the Hind III recognition site; the 7^(th) site-9^(th)site is the original initiator code);

(b) Cry j 1.1 P2 5′-GGTACCATCAACAACGTTTAGAG-3′ (SEQ ID NO:52) (the1^(st) site-6^(th) site basic group from the 5′ terminal end of theprimer sequence is the Kpn I recognition site; the 7^(th) site-9^(th)site from the 5′ terminal end of the primer sequence is the originaltermination code).

PCR fragments and pVAX1 (Invitrogen, Inc.) were subjected to thedigestion, transformation, isolation, and ligation methods as describedin Example 7, Section 2. Positive clones were selected and analyzedusing sequential analysis discussed in Example 7, Section 2 to obtainthe Cry j 1.1 expression vector, pCryj1.1.

3. pCryj1.1 Eukaryotic Expression

The pCryj1.1 expression vector was subjected to transfection, total RNAisolation, and RT-PCR protocols as previously described in Example 6,Section 3. Isolated PCR products were subjected to low melting pointagarose gel testing to confirm pCryj1.1 positive bands. The resultsprove that Cry j 1.1 may be expressed in eukaryotic cells.

Example 14 Japanese Cedar (Cryptomeria japonica) Pollen AllergenicProtein Antigen Cry j 1.2 Genetic Clone and Eukaryotic Expression Assay

1. Cry j 1.2 Primer Synthesis

The following sequences were used as Cry j 1.2 primers. Primers wereartificially synthesized

Cry j 1.2 P1 5′ primer: AAGCTTATGGATTCCCCTTGCTTAG (SEQ ID NO: 53) Cry j1.2 P2 3′ primer: GGTACCTCAACAACGTTTAGAGAGAG (SEQ ID NO: 54)2. Cry j 1.2 Expression Vector Construction

Cry j 1.2 cDNA was used as a template to perform PCR amplification usingCry j 1.2 P1 and Cry j 1.2 P2 primers to generate copies of the Cry j1.1 gene. The primers are further described below:

(a) Cry j 1.2 P1 5′-AAGCTTATGGATTCCCCTTGCTTAG-3′ (SEQ ID NO:53) (the1^(st) site-6^(th) site basic group from the 5′ terminal end of theprimer sequence is the Hind III recognition site; the 7^(th) site-9^(th)site from the 5′ terminal end of the primer sequence is the originalinitiator code);

(b) Cry j 1.2 P2 5′-GGTACCTCAACAACGTTTAGAGAGAG-3′ (SEQ ID NO:54) (the1^(st) site-6^(th) site basic group from the 5′ terminal end of theprimer sequence is the Kpn I recognition site; the 7^(th) site-9^(th)site from the 5′ terminal end of the primer sequence is the originaltermination code).

PCR fragments and pVAX1 (Invitrogen, Inc.) were subjected to thedigestion, transformation, isolation, and ligation methods as describedin Example 7, Section 2. Positive clones were selected and analyzedusing sequential analysis discussed in Example 7, Section 2 to obtainthe Cry j 1.2 expression vector, pCryj1.2.

3. pCryj1.2 Eukaryotic Expression

The pCryj1.2 expression vector was subjected to the transfection, totalRNA isolation, and RT-PCR protocols previously described in Example 6,Section 3. Isolated PCR products were subjected to low melting pointagarose gel testing to confirm pCryj1.2 positive bands. The resultsprove that Cry j 1.2 may be expressed in eukaryotic cells.

Example 15 Blomia Tropicalis Allergenic Protein Antigen Blo t 5 GeneticClone and Eukaryotic Expression Assay

1. Blo t 5 Primer Synthesis

The following sequences were used as Blo t 5 primers. Primers wereartificially synthesized

Blo t 5 P1 5′ primer: AAGCTTACAATGAAGTTCGC (SEQ ID NO: 55) Blo t 5 P2 3′primer: GGTACCAATTTTTATTGGGT (SEQ ID NO: 56)2. Blo t 5 Expression Vector Construction

Blo t 5 cDNA was used as a template to perform PCR amplification usingBlo t 5 P1 and Blo t 5 P2 primers to generate copies of the Blo t 5gene. The primers are further described below:

(a) Blo t 5 P1 5′-AAGCTTACAATGAAGTTCGC-3′ (SEQ ID NO:55) (the 1^(st)site-6^(th) site basic group from the 5′ terminal end of the primersequence is the Hind III recognition site; the 10^(th)-12^(th) site fromthe 5′ terminal end of the primer sequence is the original initiatorcode);

(b) Blo t 5 P2 5′-GGTACCAATTTTTATTGGGT-3′ (SEQ ID NO:56) (the 1^(st)site-6^(th) site basic group from the 5′ terminal end of the primersequence is the Kpn I recognition site; the 12^(th) site-14^(th) sitefrom the 5′ terminal end of the primer sequence is the originaltermination code).

PCR fragments and pVAX1 (Invitrogen, Inc.) were subjected to thedigestion, transformation, isolation, and ligation methods as describedin Example 7, Section 2. Positive clones were selected and analyzedusing sequential analysis discussed in Example 7, Section 2 to obtainthe Blo t 5 expression vector, pBlot5.

3. pBlot5Eukaryotic Expression

The pBlot5 expression vector was subjected to transfection, total RNAisolation, and RT-PCR protocols as previously described in Example 6,Section 3. Isolated PCR products were subjected to low melting pointagarose gel testing to confirm pBlot5 positive bands. The results provethat pBlot5 may be expressed in eukaryotic cells.

Example 16 Induction of Adaptive T Regulatory Cells that Suppress theAllergic Response: Conversion of T Regulatory Cells by Suboptimal DCsthat are Induced by Co-Immunization of DNA and Protein Vaccines

Total flea extracts induce immediate intradermal allergic reactions inmice, therefore this system can be used to evaluate immunotherapeuticmethods aimed at amelioration of AIH. Using this rodent model of AIHinduced by flea allergens, co-immunization of DNA and protein vaccinesencoding the flea salivary specific antigen (FSA1) amelioratesexperimental AIH, including antigen induced wheel formation, elevated Tcell proliferation, infiltration of lymphocytes and mast cells to thesite of challenge. The amelioration of AIH was directly related to theinduction of a specific population of flea antigenic specific T cellsexhibiting a CD4⁺/CD25⁻/FoxP3⁺ phenotype, a characteristic of Tr. TheseTr also express IL-10, IFN-γ and the transcriptional factor T-bet afterantigen stimulation. These Tr are driven by MHC-II⁺/CD40^(low) DCpopulations that are induced by the co-immunization of DNA and proteinvaccines. These studies identify important cellular players in thecontrol of AIH. Exploitation of these cellular regulations and theirinduction will provide novel direction to develop therapies for allergicand related disorders.

Methods

Histology analysis. On day 14 following the last immunization, skinsamples from mice were collected and fixed in 4% of paraformaldehyde,embedded in paraffin blocks from every group of mice. Four tofive-micrometer sections were cut and placed on sylan-coated glassslides prior to rehydrated in xylene and washed with decreasingconcentrations of alcohol solutions. The endogenous peroxidase activitywas blocked by 3% hydrogen peroxide at room temperature for 10 min andthe antigen retrieval was accomplished by boiling the slides in 0.01 Mcitrate buffer (pH 6.0). Lastly, slides were stained with hematoxylinand eosin (H&E) or toluidine blue for mast cells and analyzed under alight microscope for histology changes.

Skin test. On day 14 after the last immunization, the mice werechallenged with 1 μg/μl of flea-saliva-antigen on nonlesional lateralthorax skin intradermally, PBS is used as a negative control andhistamine is used as a positive control. The diameter of the skinreaction was measured within 20 min after challenge by using acalibrated micrometer. Reaction was considered as a positive when theinjection site was larger than half the size of the sums of diametersinjected comparing the positive and negative control challenges.

T cell recall responses. The T cells isolated from immunized mice on day14 were cultured at 5×10⁴ cells/well in triplicate in 96-well platescontaining RPMI-10/5% FCS and then stimulated with 20 μg/ml ofFlea-saliva-antigen for 48 h. Following the stimulation, cellproliferation was assessed by a colorimetric reaction after the additionof 20 p1 of an MTS-PMS (Pormaga, USA) solution for 2-4 hrs and its colordensity was read at 570 nm by plate reader (Magellan, Tecan AustriaGmbH).

Measurement of flea antigen-specific antibodies. Serum concentration ofanti-flea IgG1, IgG2a, IgG2b, IgM and IgE isotypes were measured usingflea antigen coated plates by ELISA and detection with specifichorseradish peroxidase-conjugated rat anti-IgG1, IgG2a, IgG2b, IgM andIgE antibodies (Southern Biotech, Birmingham, USA), absorbance (450 nm)was measured using an ELISA plate reader (Magellan, Tecan Austria GmbH).

RT-PCR. Total RNA was isolated from spleen and skin tissue 14 d afterimmunization using TRIzol reagent (Promega). cDNA was synthesized andPCR was performed in a 50 p1 reaction mixture with 5 μl cDNA and 1.0 μMof each of the following primers: HPRT, IL-2, IFN-γ, IL-4, IL-5, IL-13,IL-10 and IL-12(38). For Gata3 and T-bet analysis, CD4⁺CD25⁻ T cellswere isolated and total RNA was prepared. RT-PCR was done as describedusing specific primer sequences as follows forGata3,5′-GGAGGCATCCAGACCCGAAAC-3′ (forward) (SEQ ID NO: 57) and5′-ACCATGGCGGTGACC-ATGC-3′ (reverse) (SEQ ID NO: 58); for T-bet,5′-TGAAGCCCACACTCCTACCC-3′ (forward) (SEQ ID NO: 59); and5′-GCGGCATTTTCTCAGTTGGG-3′ (reverse) (SEQ ID NO: 60).

Isolation of CD4⁺CD25″T cells and adoptive transfer. Single splenocytesuspensions were prepared from mouse spleen and CD4⁺CD25⁻ T cells wereisolated and purified by using the MagCellect Mouse CD4⁺CD25⁺ RegulatoryT Cell Isolation Kit according to the manufacturer's protocol (R&DSystems, Inc., USA). The purity of the selected cell populations was96-98%. The purified cells (1×10⁶ per mouse) were adoptively transferredintravenously into C571BL6 mice.

CFSE labeling and co-culture of cells. Naive CD4⁺ T cells isolated fromC57/BL6 mice were labeled with CFSE (Molecular Probes). For assay ofregulatory activity, 1×10⁴ pcDF100+F induced regulatory or control Tcells were co-cultured with 4×104 purified and CFSE labeled naive CD4⁺ Tcells in the presence of flea antigen (100 μg/ml) and 1×104 bone marrowderived DCs. For some cultures, Tr cells were co-cultured withanti-IL-10, anti-IFN-γ or an isotype control antibody at 100 g/ml. After72 h, cells were collected and labeled then CFSE+ cells were selectedfor analysis by flow cytometry.

Flow cytometry. CD4⁺CD25⁻ T cells were isolated and incubated on icewith PE-conjugated antibodies to CD44, CD69, CD62L (eBioscience, Calif.,USA). Flow cytometry of cytokine production and FoxP3 expression in Tcells were performed, single cell suspensions were prepared from theanimals spleens and Fc receptors were blocked with excess anti-Fc (BDPharMingen, USA). Cells were washed with ice-cold PBS. For intracellularcytokine staining, T cells were stimulated overnight with Con A(Sigma-Aldrich) in the presence of anti-CD28 mAb (BD PharMingen, USA).Collected cells were fixed with 4% paraformaldehyde and permeabilizedwith 0.1% saponin (Sigma-Aldrich). For staining of surface of CD4 orcytoplasmic IL-10, IL-4, IFN-γ or FoxP3, the appropriate concentrationsof phycoerythrin-labeled antibodies (eBioscience, Calif., USA) wereadded to premeabilized cells for 30 min on ice followed by washing twicewith cold PBS. Samples were processed and screened using FACSCalibur anddata were analyzed with Cell Questpro software (BD).

Results

A flea salivary allergen, FSA1, has been identified and implicated asone of the causes for allergy dermatitis observed in cats and dogs. Thedegree of skin reaction or intradermal test (IDT) to assess theimmediate intradermal flea-antigen reactivity can be achieved by fleaallergy challenge intradermally. As expected, administration of micewith flea antigen induced significant skin reactions (FIG. 1 a), inducedmast cells activation, induced coincident IgE production (FIG. 1 b), andinduced strong CD4+ T cell proliferation responses (FIG. 1 c) in C57mice when compared with naive control animals after intradermalchallenge. This data demonstrates the utility of the flea antigenallergic model for evaluation of novel therapeutic strategies againstAIH.

This model was used to examine the ability of the co-inoculationstrategy to protect animals from the immediate hypersensitivity afterflea allergen challenge. C57/BL6 mice were pre-sensitized with varioustest vaccines and animals were intradermal challenged with the fleaextracts, or with histamine as the positive or using PBS as the negativecontrol. The immediate hypersensitivity reaction was blocked in groupimmunized 14 days earlier of co-inoculation of a plasmid DNA, pcDF100,encoding an epitope of FSA1 (aa100-114) mixed with the total fleaproteins (designated as pcDF100+F) (FIG. 2 a). To determine if theinhibition observed was due to the plasmid backbone or rather wasrelated to a DNA construct encoding another region of FSA1. Robustinflammation of the challenged sites were observed in the mice immunizedwith either vector control plus flea proteins (designated as V+F), or aDNA construct encoding another region of FSA1 at aa66-80, pD66, againmixed with the flea proteins (designated as pcDF66+F, FIG. 2 a). We alsoexamined the influence of host immune balance toward Th1 type on theinhibition of allergic reaction, the pcDF100 primed and the flea proteinboosted animals (designated as pcDF100→F; FIG. 2 a). The severe reactionfollowing challenge was observed in mice primed with pcDF100 and boostedwith F (FIG. 2 a), suggesting that induction of a strong immune responseworsens the allergic reaction.

Histological analysis revealed infiltrations by leukocytes and mastcells in the skin lesions of mice immunized with F or V+F at thechallenge sites; whereas, mice immunized with pcDF100+F showed normalintradermal structure which was free of inflammatory cells.

We next analyzed if the blocking of the immune reaction induced by theco-inoculation was a dose dependent. pcDF100 at dose of 25, 50, 100 and200 μg was co-immunized with 100 μg of flea proteins, respectively.Dosage at 50 μg of pcDF100 showed significant inhibition of theintradermal reaction which reached maximal inhibition at 100 μg. Adosage of 25 μg exhibited only a minimal effect on lesion formation, asthe animals developed severe reactions similar to those observed inanimals inoculated with either F or V+F (FIG. 2 b) or positive controls.

High levels of IL-4, IL-5 and IL-13 are the characteristics of allergicreactions and these immune modulators are implicated in allergyseverity. Different profiles of the cytokines associated with theco-inoculated regimens were examined. Mice co-inoculated with F or V+Fproduced higher level of mRNA expressions for IL-4, IL-5 and IL-13;whereas, mice co-immunized with pcDF100+F produced relative low levelsof these cytokines, suggesting that an anti-inflammatory immuneregulatory function was derived from the co-inoculation of pcDF100+F. Nosignificant differences were found in the levels of IL-2 or IFN-γ amongthe co-inoculated regimens, but IFN-γ was slightly higher in mice primedwith pcDF100 and boosted by F. This result again suggests that theinduced inhibition of the allergic reaction by co-immunization is notdue to an un-balanced Th2 to Th1 response by the allergenic-specificT-helper cells.

As flea antigen triggered IgE-mediated allergy is well characterized,the ability of co-inoculation of pcDF100+F to inhibit anti-flea inducedIgE production was examined. The levels of anti-flea IgE and IgG in serawere measured on days 14, 28 and 42 and were reduced slightly in miceco-immunized with pcDF100+F compared with groups immunized with V+F or Falone (FIG. 2 c), suggesting that the co-inoculation does not influencethe IgE production.

Proliferative CD4⁺ T cells are known to be involved in the developmentof immediate hypersensitivity. I isolated CD4⁺ T cells from the spleenof mice co-immunized with F, V+F or pcDF100+F was examined on day 14after the second immunization for their recall proliferative responsesto flea antigens in vitro. Immunizations of F and V+F resulted in strongproliferation of CD4⁺ T cells; whereas the CD4⁺ T cells isolated frompcDF100+F immunized mice showed little, if any, proliferation inresponse to the flea antigen stimulation (FIG. 2 d). These resultssuggest that the inhibition of hypersensitivity observed byco-immunization of pcDF100+F is related to the non-responsive antigenspecific CD4⁺ T cells.

The results indicate that the co-immunization with pcDF100 and fleaproteins antigen induces an inhibition of the allergic reaction viadown-regulating the levels of inflammatory cytokines and CD4⁺ cellsinduced by flea intradermal challenge. This suggests that the preventionof allergy is likely antigen specific since the antigen mismatchedcombinations did not produce the same effect.

To test if antigen-specific Tr cells have been induced by theco-immunization of flea DNA and protein vaccines, splenocytes werecollected from co-immunized C57B/6 mice and mixed with theflea-antigen-specific effector T cells of syngeneic mice to exam theirability to inhibit recall proliferative responses in vitro. As shown inFIG. 3 a, splenocytes from mice co-immunized with pcDF100+Fsignificantly inhibited T cell recall immune responses. In contrast,splenocytes from mice immunized with F or V+F as well as from naive micefailed to inhibit the antigen specific T cell proliferative responses(FIG. 3 a). This indicates that cells within the splenocyte populationlikely generated during the co-immunization of animals can suppress thisantigen specific T cell proliferation. Purified non-T cells, T cells orsubsets of T cells from the spleen of mice co-immunized with pcDF100+Fwere identified and tested individually for suppression of recallproliferation. Significant inhibition was observed from reactions ofeither purified T cells, purified CD4⁺ cells, or purified CD4⁺CD25″cells from the mice co-immunized with pcDF100+F, but not from othersubsets of cells (FIG. 3 a). However, the inhibitions from CD4⁺CD25⁺cells are in general thought to be independent of antigen sensitization,whereas the inhibition of CD4⁺CD25″ cells of this reaction is in anantigen dependent manner (FIG. 3 a).

To further examine this issue in vivo, adoptive transfer was utilized.Antigen naive syngeneic recipient mice were adoptively transferred witheither total splenocytes, T, CD4⁺ or CD8⁺ cells isolated from C57B/6mice co-immunized with pcDF100+F, F or V+F, respectively. All recipientmice were next challenged intradermally by flea extract to induce thehypersensitivity. Splenocytes, T and CD4⁺ T cells from mice immunizedwith the pcDF100+F, but not with the F or V+F, were all able to suppressthe development of the immediate hypersensitivity reaction (FIG. 3 b).In contrast, CD8⁺ T cells isolated from all three experimental groupsand naive control mice did not suppress this reaction. Both in vitro andin vivo results indicate that CD4⁺CD25⁻ Tr cells can mediate thissuppression.

To investigate the observed role of CD4⁺CD25⁻Tr antigen specificity, theCD4⁺C25⁻ Tr cells taken from C57B/6 mice co-immunized with pcDF100+Fwere adoptively transferred into the syngeneic recipient mice that weresubsequently immunized twice at a biweekly interval with flea-antigen orOVA in Freunds' complete adjuvant (FCA). On day 14 after the lastimmunization, T cells were isolated and tested for their ability toproliferate to either flea antigen or OVA in vitro. T cells fromrecipient mice immunized with flea antigen did not respond the fleaantigen stimulation in vitro; whereas, the T cells from recipient miceimmunized with OVA-FCA responded well with OVA stimulation, but not tothe flea antigens stimulation in vitro. As the controls, the naive miceimmunized with flea antigen respond well to the flea antigenstimulation, but not to OVA stimulation in vitro and vice versa (FIG. 3c). This result indicates that adoptive transferred CD4⁺C25⁻ Tr cellsonly inhibit the flea antigen specific T cell priming and proliferationin vivo; while the responses from the irrelevant antigen specific Tcells were not affected.

Taken together, these data demonstrate that CD4⁺CD25⁻ Tr cells wereinduced by the co-immunization of DNA and protein vaccines. These appearto be unique CD4+ Tr cells as they function in an antigen-specificmanner.

To determine if Tr cells induced by co-inoculation express certain typesof cytokines and unique markers associated with Tr cells as previouslydocumented (J. D. Fontenot, M. A. Gavin, A. Y. Rudensky, Nat Immunol 4,330 (Apr. 1, 2003); M. G. Roncarolo, R. Bacchetta, C. Bordignon, S.Narula, M. K. Levings, Immunol Rev 182, 68 (Aug. 1, 2001); and P. Stocket al., Nat Immunol 5, 1149 (Nov. 1, 2004)), CD4⁺CD25⁻ cells wereisolated from the mice immunized with F, V+F or pcDF100+F on days 1, 3,7 and 14 post immunization and T cell profiles were followed byintracellular staining with specific fluorescent labeled antibodies.After co-immunization, on days 1, 3, 7 and 14, CD4⁺CD25⁻ T cells wereisolated from F, V+F and pcDF100+F immunized mice and intracellularcytokine production for IL-10, IFN-γ and IL-4 expression was assessed byflow cytometry. CD4⁺CD25⁻ Tr cells isolated from F, V+F, pcDF100+F andnaive mice as controls were analyzed for expression of CD69, CD44 andCD62L, and for their expression of FoxP3. Tr cells express T-bet but notgata-3. On day 14 after immunization, total RNA was extracted fromCD4⁺CD25⁻ T cells from all three groups and RT-PCR was used to test theexpression of HPRT, T-bet and gata-3. Over the course of 14 days, theCD4⁺CD25″T cells isolated from mice co-immunized with pcDF100+Fexpressed high levels of IL-10, IFN-γ, FoxP3, and a minimal amount ofIL-4. In contrast, CD4⁺CD25″T cells produced a higher level of IL-4 andno expressions of FoxP3, IL-10, or IFN-γ from the mice immunized with For V+F. Since the transcriptional factor Foxp3, has been demonstrated tobe a hallmark of the Tr cells, the co-vaccination induced CD4⁺CD25⁻ Trcells can be categorized into the regulatory class of T cells but theyhave a unique phenotype. T cell activation markers are expressed equallyhigh including CD44 and CD69 and low for CD62L among the immunizedgroups, suggesting that the induced CD4⁺CD25⁻ Tr cells are fullyactivated by the immunization.

To analyze the Th phenotype for the induced CD4⁺CD25⁻ Tr cells based onthe observed cytokine expression patterns as described above, theexpression of both T-bet and gata-3 genes of the CD4⁺CD25⁻ T cells frommice immunized with F, V+F or pcDF100+F were analyzed by the RT-PCRmethod. The results show that the CD4⁺CD25⁻ T cells from the pcDF100+Fimmunized mice, but not from the F or V+F immunized mice, expressedhigher level of T-bet, a hallmark for Th1 cells. In the contrast, theCD4⁺CD25⁻ T cells from the F and V+F immunized mice expressed higherlevels of gata-3, a characteristic Th2 cells.

Collectively, these data demonstrate that CD4⁺CD25⁻ T cell induced bythe co-immunization of DNA and protein vaccines has as an adaptive Th1phenotypic Tr cell which can suppress the antigen-specific CD4⁺ T cellproliferative function.

Since antigen presenting cell (APC) activates T cells to promoteadaptive immunity, the induction of CD4⁺CD25⁻ Tr cell is apparentlythrough specific APC activation by the co-inoculation of DNA+proteinvaccines. To investigate this question, a similar experiments describedas above was set up to assess dendritic cell (DC) function and phenotypeand their influence on naive T cells. The effects of co-inoculation onthe maturation of DC was analyzed. Expression of costimulatory moleculeson DCs from co-immunized mice was examined. Splenocytes were isolatedand stained for expression of surface markers on DCs by gating on CD11positive cells 48 h after co-immunization of pcDF100+F, V+F or F.Co-immunization of pcDF100+F did not affect maturation of DCs as DCsisolated from the spleen 48 h after the co-immunization expressed highand similar levels of CD80, CD86, MHC II, IL-12, IL-6, IL-1a/f3, IFN-a/8and TNF-a which are the characteristics of matured DCs; whereas thesemolecules on the immature DCs from naive control mice remain expressedat relatively low levels, suggesting the co-inoculation enables theimmature DC be induced to mature. However, we observed that the level ofCD40 expression was dramatically reduced in pcDF100+F co-immunized micecompared to all other groups, suggesting a unique phenotype of DC may beinvolved in the induction of the observed Tr. DCs from mice immunizedwith V+F or F were observed to have the ability to activateheterogeneous T cells to proliferate; whereas the Immature DCs of naivemice have no such ability as expected (FIG. 4 a). Interestingly, DCsfrom mice co-immunized with pcDF100+F had a restricted capacity toactivate T cells to proliferate (FIG. 4 b), suggesting that analternative mechanism of Tr induction is induced in the co-immunizationgroup. To explore if the DC of co-immunized mice are able to convertnaive T cells to a Tr phenotype, DCs obtained from mice after theirbeing co-immunized with pcDF100+F were co-cultured with syngeneic naiveCD4⁺ T cells in vitro and subsequently characterized the resulting CD4⁺T cells by FACS. These T cells upregulated higher levels of CD44, andCD69, but lower levels of CD62L, suggesting that the DC again has thecapacity to activate T cells. Similar results were obtained fromanalysis of DCs from V+F or F co-immunized mice. Furthermore, theactivated CD4⁺ T cells in the pcDF100+F group produced significanthigher level of IL-70 and IFN-γ, but reduced IL-4 (FIG. 4 b). However,activated T cells from V+F or F immunized groups produce significantlevel of IL-4, but little IL-10 and IFN-γ (FIG. 4 b). The cytokineproduction were analyzed after three rounds of re-stimulation with freshDCs isolated from the D+F immunized mice. These studies demonstrated anincrease in the production of IL-10 in T-cells, which is one ofcharacteristic of regulatory T cells. To further characterize theirregulatory function, IL-10⁺ T cells were isolated after DC stimulationand subjected to MLR to see if they block responder T cell proliferationin the MLR. As shown, the proliferation of responder T cells wereinhibited by the presence of IL-10⁺ T cells, but not from T cellsisolated from co-culture with DCs of mice immunized with V+F and F, orthe control animals (FIG. 4 c). These data demonstrate that DCs from theD+F co-immunized mice develop large populations of T regulatory cells invitro.

To demonstrate the same conversion in vivo, DCs collected from BALB/cmice after co-immunization with pcDF100+F were co-transferred withsyngeneic naive CD4⁺ T cells into nude mice (nu/nu) and subsequentlyanalyzed these transferred T cells by FACS for intracellular stainingfor IL-10 and T reg markers. DCs from pcDF100+F co-immunized miceinduced Tr cells in vivo. DCs isolated from spleens of pcDF100+F, V+F, Fimmunized or naive control mice were co-adoptive-transfer with naiveCD4⁺CD25⁻T cells. The T cells were analyzed for IL-10, IFN-γ, IL-4,FoxP3 and CD25 on day 3 and 7. Co-transferred T cells expressed moreIL-10, FoxP3 and IFN-γ, but little IL-4. However, co-transferred T cellswith DCs from V+F or F immunized mice expressed higher levels of IL-4,but little IL-10 and IFN-γ, supporting the in vitro data. Specific IL-10cytokine production was elevated after two rounds of re-stimulation withthe DCs freshly isolated from D+F immunized mice. These data demonstratethat DCs from D+F co-immunized mice drive likely naive T cells into Trcells in vivo. Consistent with previous results, such conversion wasonly made within the CD4⁺CD25″ population since the frequency of CD25⁺was not observed to be influenced testing vivo.

Finally, experiments were performed to identify what molecules in theinteraction between DC and T cells play a role in the development of Tr.Since the Tr is within the activated T cell compartment as demonstratedabove, the signals should include classic activation pathway signals.Important among these is the up-regulation of major histocompatibilitycomplex (MHC) class II and co-stimulatory molecules (CD80/CD86), whichprovide the two requisite signals for naive T cell activation. To studythis issue, DCs were isolated from spleen of mice 48 h after pcDF100+Fco-immunization and co-cultured with naive CD4⁺ T cells from naivesyngeneic mice in the presence or absence of reagents to block signalingmolecules including anti-CD80, anti-CD86, anti-CD40 and anti-MHC-II.After seven days of re-stimulation, T cells were isolated and added intoMLR system to examine their regulatory functions. The results showedthat both anti-CD40 and anti-MHC II mAbs can partially reverse thesuppressive effects on MLR by the Tr cells; whereas mAbs against CD80,CD86 and had no ability to block the induction of the suppressionphenotype. These results demonstrate that both CD40 and MHC II signalsplay roles in the induction of Tr in the flea allergen Induced immediatehypersensitivity model.

Adaptive Tr1 cells have been observed and induced by DCs processing asuboptimal immunogen or subimmunogen to inhibit normal T cells' functionmediated via secretion of IL-10 or TGF-b or both. It has been furtherdemonstrated that immature DCs drive the differentiation ofIL-10—producing Tr1 cells with producing IL-10, TGF-R, and IFN-γ, butthey do not produce IL-4 or IL-2, they are hyporesponsive toantigenspecific and polyclonal activation. In additional evidence hasshown that suboptimal activation of DC by a minute antigen stimulationcan induces the Tr1 conversion. This stimulation of DCs lacks theco-stimulation signaling and thus presents a tolerance signal to Tcells.

Co-inoculation of DNA encoding a flea antigen with flea protein inducesadaptive Tr cells which inhibit the allergic reaction induced by fleaallergen challenge. The cells exhibit a phenotype of CD4⁺CD25″Foxp3⁺ andsuppress in vivo and in vitro antigen specific as well as MLR immuneresponses through the production of IL-10 and IFN-γ. MHC-11+/CD40^(low)DC populations are induced by such co-immunization and in turn toconvert naive T cells into Tr cells.

Example 17

Testing the Prevention and/or Therapeutic Approaches Against FADAllergen Through Co-Immunization with Vaccines in a Feline FAD ModelEstablishing the Feline Model and Testing Co-Immunization

There are more than 15 kinds of allergen in the flea saliva. Among them,an 18 kDa protein, flea saliva antigen 1 (FSA1), has been determined amain allergen which can cause FAD. This gene has been cloned andexpressed it in the E. coli system. In addition, the pVAX-FSA1eukaryotic expressing construct has also been prepared.

A cat FAD model was established and subsequently used to demonstratethat the therapeutic effect of co-immunization of FSA1+pVAX-FSA1 on theestablished FAD in cats.

Determination of Number of Fleas, Duration of a Cycle Animal andParasite

Ten pathogen-free cats were purchased from North China PharmaceuticalGroup (Shijiazhuang, Hebei) and housed animal facility at the Center forDisease Control and Prevention of China (CDC) in the course ofexperiment. All cats were over one year old, which is an importantfactor since the younger animals may be tolerable to the flea allergenin this experiment. The cats were grouped with breed and sex randomly.The sterile fleas were supplied by the CDC.

To determine relationship of infestation and FAD symptom, 10 cats wereseparated into three groups, including an experimental group with sixcats and two control groups with two cats each. One control group wastreated nitenpyram, and another was not treated with the nitenpyram.Each experimental cat was lived separated on day 0 and infested with 100fleas. After two days, all cats in this group were given the nitenpyramto remove the flea from their bodies. Two of the control cats were alsogiven this medicine on day 0. This challenge cycle was repeated everyother week for 7 times

Immunization Scheme for the FAD Induced or Control Cats With FSA1 andpVAX-FSA1 as Co-Immunogens

The 6 FAD cats were separated into three groups. Two of them wereco-immunized with 400 μg FSA1 i.p. and 400 μg pVAX-FSA1 plasmid atdouble lateral abdomen subcutaneously. Two were co-immunized with 400 μgFSA1 i.p. and 400 μg pVAX vector subcutaneously. The last two were notimmunized and used as the positive control group. The 4 un-FAD cats wereseparated into two groups. Two were co-immunized with 400 μg FSA1 i.p.and 400 μg pVAX-FSA1 plasmid at double lateral abdomen subcutaneouslyand the other two were immunized with 400 μg FSA1 i.p. and 400 μg pVAXvector subcutaneously. The two control cats with nitenpyram were sentinto different groups. The cats were immunized three times: at days 0,9, and 16. After that, the cats were challenged for six cycles as above.During the second immunization, a cycle of challenge was done at thesame time, because we wanted to keep positive cats at the susceptiblestate for next therapy. The program of immunization was as Table 2.

TABLE 2 Dermatological scores. Immunization PSA1 + PSA1 + pVAX Treatwith Cat No. pVAX-FSA1 vector nitenpyram prophylactive 1 − + + 2 + − −4 + − + 5 − + − therapeutic 8 + − + 9 − + + 10 − + + 11 + − + Positive 3− − + control 7 − − +Method

Cats were scored by the dermatological assessments two days afterceasing each infestation. The assessments were included erythema,papules, crusts, scale, alopecia, excoriation. The body of each cat wasdivided into three portions to assess which part might have the mostsevere clinical outcome. According the previous documented report, threeparts were consisted of: (1) back, from the head to the tail of dorsalsurface; (2) double lateral abdomen, from the scapula to the tail; (3)chest and underside, from laryngeal to the caudomedial thighs.

Smearing Counts of Peripheral Blood Cells from the Cats Method:

The anti-coagulant peripheral blood at 2 ml was collected on days 0, 2,16, 30, 44, 58, 62 from the experiment group and day 0, 14, 28, 42, 56,60 from the control groups. The same samples were collected after theimmunization. A drop of blood was smeared on a clean glass slide. Afterthe smear was dried, the cells were stained with Wright-Giemsa stain for15 min. After a rinse in deionized water, the smear was gently driedwith Kimwipe paper and dehydrated by 96% and 100% ethonol with each 10seconds treatment. The slide was then treated with xylene for 30 min.

After the staining, nucleus and cytoplasm of blood cell wasdistinguished by a blue and pink staining. The percentage of each typeof cells was counted by a total number of 200 cells in a view under alight microscope.

Therapeutic Approaches:

The same methods as above were used except that total RNA was extractedfrom mononuclear cells which were isolated from peripheral blood on day7 post the third immunization from each group.

Statistical Analysis

Analysis of variance (ANOVA) was used to detect the differences whichincluded dermatology scores from each cycle, scores of various lesionsand scores on different sites among three groups. The differences ofskin test and IgE level were determined also by ANOVA. Statisticalanalyses of other ones were performed using the Student's t-test. Inthese analyses, data were converted into logarithmic plot. If theP<0.05, indicates a significant difference.

Statistical analyses of were also performed using the Student's t-test.In these analyses, the data was converted into log. If the P<0.05, thedata indicated significant differences.

Results:

Observation and Dermatological Scores of the Cats

Lesions were scored according to their types, locations, sizes andnumbers.

The total scores were analyzed from the groups, and the lesions andsites for the experimental group were assessed. The FAD group registeredmore scores than in other groups. The scores were increased at thefourth infestation cycle, and then stayed at the level of 5.0. Thescores in the FAD group were significant higher than in other controlgroups (P<0.01). These results support the conclusion that the cat FADmodel was valid and feasible to evaluate a therapeutic or prophylactictreatment against the allergic reaction.

To assess where the lesions occur most, what type, and when to occur,statistic analysis was done and it was found that papules and fur losswere the most common factors to contribute to the dermatology scores inFAD group. The reading of erythema was not a contributing factor sincethe erythema reached to the peak on day 44, but it fallen at the end ofthis experiment, indicating no persistency. On the other hand, thepapules were increased, and remained at a high level after 44 days andreached to the peak on the day 86 (P<0.05), suggesting its persistency.Similarly, fur loss was maintained at high level after day 44 (P<0.05).These two readouts are indicated as a good onset to reflect FAD. Thescores from other lesions were randomly distributed and inconsistent inthe two control groups, in which no significant difference of lesionswere observed.

Although, most of dermal lesions tended to be located on the backs andheads compared other sites on day 44 (P<0.05), the lesions were extendedall over the body at the end of the experiment. This observation wasdifferent from the previous reports in dog, of which the chest andunderside were tended to have the most lesions. That may owe to thehabitual differences of the cats and dogs.

To eliminate the interference by the nitenpyram treatment, anydifferences between these treated with nitenpyram and the ones withouttreatment were assessed. From the dermatology score, no significantdifference was seen in the control groups. Thus, the nitenpyramtreatment did not influence the results obtained from our experiments(FIG. 5).

Cell Percentage in Peripheral Blood by Blood Smear Analysis

A comparison of the number of each type of cells in peripheral bloodfrom the different groups showed that the number of eosinophils washigher in the experimental group than those in control groups. Bloodsmears were performed for each infestation cycle, but the percent ofeach type of cells became constant after the third cycle. As the resultfrom day 58 show in Table 3, the significant changes occurred in thenumber of eosinophils among the three groups. Since the increase ofeosinophils is related to allergic diseases as previously documented,these results indicate that the cats in the experimental group were moresusceptible to the infestation of fleas. There was no obvious differencebetween the two control groups, the nitenpyram seems not to interferethe cell numbers in the cats' peripheral blood.

TABLE 3 Cell types and their percentage in peripheral blood LymphocytesMonocytes Neutrophils Basophils Eosinophils (%) (%) (%) (%) (%)Experimental group 23.4 2.6 61.5 1.0 11.5 Control group with nitenpyram21.8 3.6 70.5 0.7 3.4 Control group without nitenpyram 23.1 3.6 68.3 0.64.4Histopathological Examination

First, the differences between the normal skins and those with lesionswere analyzed. Skin biopsies were chosen from all groups on day 58.However, the skin biopsies from one cat may also display differences insome degree, especially those with lesions. That was not enough toindicate whether the cats infested with fleas were induced to have FAD.For this reason, the skin biopsies from each group were collected afterthe IDT with the flea extracts since IDT with flea provides an antigenspecific recalled immune responses.

Skin Test to Evaluate Whether the Cats were Allergic to the FleaExtracts.

The diameter of wheal or bleb was measured 15 min after the IDTinjection. Since each cat had a different level of sensitivity to theIDT, the results from each individual animal were recorded. FIG. 6 showsthe skin reactions in all groups. An allergic reaction was considered tohave occurred if an IDT value was above the average of threshold (thesum of saline and histamine is divided by 2), or not occurred if an IDTvalue was below the average of the threshold. The results are shown inTable 4. Cats with dermatology scores at 5.0 or above were identified asthe positive for FAD.

TABLE 4 Comparison of dermatological assessment with the intradermalskin test (IDT). Control Positive Positive Positive Control groupPositive skin test skin test Positive skin test Cat Experimental groupwith without Dermatology clinical with with flea skin test with No.group nitenpyram nitenpyram scores scores BSA extract with FS FSA1 1 NoYes No 0 − − − − − 2 No No Yes 1 − − − − − 4 No Yes No 1 − − − − − 5 NoNo Yes 0 − − − − − 3 Yes No No 7 + − + − + 7 Yes No No 6 + − + − − 8 YesNo No 8 + − + − + 9 Yes No No 2 − − + − + 10 Yes No No 6 + − + − + 11Yes No No 3 − − + − +Skin Test to Determine the Effect of Co-Immunization of FSA1 andpVAX-FSA1

The cats with FAD were co-immunized with FSA1+pVAX-FSA1 or vector+FSA1as described in experimental design B in Table 2. Before and after theimmunizations, the cats were tested by IDT with various flea antigens orcontrol antigens. The IDT readings were recorded on 7 days after thelast immunization as summarized in FIG. 7 and Table 5.

The results showed that FAD cats co-immunized with the FSA1+pVAX-FSA1had less skin-reactions to the flea extracts or flea specific antigens(such as FSA1 protein) challenges; whereas the cats immunized withvector+FSA1 had more skin-reactions to the same challenges.

Comparing the differences before and after immunization, we observedthat the skin reaction was much smaller after the immunization thanbefore in the co-immunized group as shown FIG. 7, suggesting theco-immunization significant decreased sensitivity to the flea challenge.On contrary, no significant effect was seen in groups immunized withFSA1 and pVAX vector, indicating that the cats were remained theirallergic status. The status of all cats was listed in Table 5.

TABLE 5 The state IDT for FE FSA1 FSA1 + IDT with IDT before CatPositive and pVAX- with flea with immuni- No. control pVAX FSA1 BSAextracts FSA1 zation 1 No Yes No − 2 No No Yes − 4 No No Yes − 5 No YesNo − 3 Yes No No − + − + 7 Yes No No − + − + 8 No No Yes − − − + 9 NoYes No − + − + 10 No Yes No − + − + 11 No No Yes − − − +Therapeutic Effects of Co-Immunization on the FAD Cats

To determine the therapeutic effect of co-immunization on lesions of theFAD cats, the lesions were recorded over period of 53 days after thefirst immunization and shown in FIG. 8. The lesion scores in theco-immunized group were dramatically reduced from 5.5 to 2 (FIG. 8,square points on line). Whereas, the effects on the group immunized withFSA1 and pVAX vector was reduced but to a lesser extent (FIG. 8,triangle points on line). The result suggested the co-immunization had atherapeutic effect on the established FAD in cats.

Correlation of the Type of Lesions Affected by the Co-Immunization onthe FAD Cats

To correlate which type of lesions reduced with the co-immunization, thescores on each type of lesions in every group were analyzed and theresults are shown in FIGS. 9A-9F. Only the score of the papules werereduced from 4.0 to 0.5 and coincident with the co-immunization ofFSA1+pVAX-FSA1 (FIG. 9B, square points on line). Other type of lesionsin experimental group was remained unchanged.

Correlation of the Location of Lesions Affected by the Co-Immunizationon the FAD Cats Therapeutic Effects of Co-Immunization on the FAD Cats

After analysis the correlation, the data showed that the scores ofdouble lateral abdomen in therapeutic group were reduced from 1.5 to 0(FIG. 10B) after the co-immunization of FSA1+pVAX-FSA1, but no effect inthe other places as shown in FIGS. 10A-10C. The therapeutic groupimmunized with FSA1 and pVAX vector showed a lingering response (FIG.10B, triangle points). The dermatologic scores of FAD cats immunizedwith FSA1 and pVAX vector were reduced from 1.5 to 0.5 after 7 days ofthe last immunization. In contrast, those in the co-immunized withFSA1+pVAX-FSA1 were reduced promptly after its first immunization andremained at low level thorough (FIG. 10B, square points).

Summary

These data demonstrate the successful induction of a FAD model in felineby flea infestations. Physiological or pathogenic parameters in the FADfeline have been characterized which can be used as a model to evaluatetreatment of immunotherapeutic or prophylactic approaches.

The co-immunization of FSA1+pVAX-FSA1 vaccines was demonstrated tosuppress the established FAD in cats. Such suppression seems to be anantigenic specific, which supports the results observed in mousestudies.

Example 18 pVAX1-K-FSA1

Plasmid pVAX1-K-FSA1 comprises the FSA1 coding sequence linked to aKozak sequence in plasmid backbone pVAX (Invitrogen). The sequence ofthe K-FSA1 insert is SEQ ID NO:61. Nucleotides 1-9 correspond to theKozak sequence. the open reading from of FSA1 plus missed 8 amino acidsbegins at nucleotide 10. A map of the plasmid pVAX1-K-FSA1 is shown inFIG. 11.

1. A therapeutic composition for inhibiting an allergic response against a flea allergenic protein comprising: a) an eukaryotic cell expression vector comprising a nucleotide sequence that encodes an amino acid sequence comprising SEQ ID NO. 2; and b) a protein or polypeptide encoded by said nucleotide sequence.
 2. The therapeutic composition of claim 1, wherein the nucleotide sequence encodes an amino acid sequence consisting of SEQ ID NO.
 2. 3. The therapeutic composition of claim 1 wherein the eukaryotic cell expression vector comprising the nucleotide sequence encoding an amino acid sequence comprising the sequence of SEQ ID NOs: 2 is operably linked to a promoter selected from the group consisting of RSV, CMV, and SV40 promoters.
 4. The therapeutic composition of claim 1 wherein the ratio of amount of eukaryotic cell expression vector by weight to amount of the protein or the polypeptide encoded by said nucleotide sequence by weight is between 1:5 and 5:1.
 5. The therapeutic composition of claim 1 wherein the ratio of amount of eukaryotic cell expression vector by weight to amount of the protein or the polypeptide encoded by said nucleotide sequence by weight is 1:1.
 6. The therapeutic composition of claim 1 wherein the molar ratio of eukaryotic cell expression vector to the protein or the polypeptide encoded by said nucleotide sequence is between 1:100,000 to 20:100,000.
 7. The therapeutic composition of claim 1 wherein the molar ratio of eukaryotic cell expression vector to the protein or the polypeptide encoded by said nucleotide sequence is 15:100,000.
 8. A kit for inhibiting an allergic response against a flea allergenic protein comprising: a) a first container comprising an eukaryotic cell expression vector comprising a nucleotide sequence encoding an amino acid sequence comprising SEQ ID NOs: 2; and b) a second container comprising a protein or polypeptide encoded by said nucleotide sequence.
 9. The kit of claim 8 wherein the eukaryotic cell expression vector comprising the nucleotide sequence encoding an amino acid sequence comprising the sequence of SEQ ID NOs: 2 is operably linked to a promoter selected from the group consisting of RSV, CMV, and SV40 promoters.
 10. The kit of claim 8 wherein the ratio of amount of the eukaryotic cell expression vector by weight to amount of the protein or the polypeptide encoded by said nucleotide sequence by weight is between 1:5 and 5:1.
 11. The kit of claim 8 wherein the ratio of eukaryotic cell expression vector by weight to amount of the protein or the polypeptide encoded by said nucleotide sequence by weight is 1:1.
 12. The kit of claim 8 wherein the molar ratio of eukaryotic cell expression vector to the protein or the polypeptide encoded by said nucleotide sequence is between 1:100,000 to 20:100,000.
 13. The kit of claim 8 wherein the molar ratio of eukaryotic cell expression vector to the protein or the polypeptide encoded by said nucleotide sequence is 15:100,000. 